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CN103402525A - Methods of inhibition of protein fucosylation in vivo using fucose analogs - Google Patents

Methods of inhibition of protein fucosylation in vivo using fucose analogs Download PDF

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CN103402525A
CN103402525A CN2011800383342A CN201180038334A CN103402525A CN 103402525 A CN103402525 A CN 103402525A CN 2011800383342 A CN2011800383342 A CN 2011800383342A CN 201180038334 A CN201180038334 A CN 201180038334A CN 103402525 A CN103402525 A CN 103402525A
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alkyl
aryl
heterocycle
fucose
halogen
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CN103402525B (en
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P·森特
S·阿利
D·本杰明
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Seagen Inc
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Seattle Genetics Inc
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Abstract

The invention provides methods and compositions for the inhibition of fucosylation of proteins, including antibodies, in vivo by administration of a fucose analog.

Description

Use the fucose analog method of Profilin matter fucosylation in vivo
The cross reference of related application
The application requires the U.S. Provisional Patent Application of submitting on August 5th, 2010 number 61/371,116, priority, the disclosure of this provisional application is included this paper by reference in.
Background technology
L-fucose is a kind of monosaccharide, has another name called L-fucose, is some N-connecting-types in animal and the component of O-connecting-type polysaccharide and glycolipid.(seeing Becker and Lowe, Glycobiology13:41R-51R (2003) .) fucose, usually used as the end modified polysaccharide that is added into, comprises blood group antigen, selects element and polysaccharide that antibody connects.Fucose can by the specificity fucosyltransferase through α (1,2)-, α (1,3)-, α (Isosorbide-5-Nitrae)-be incorporated on polysaccharide with α (1,6)-johning knot.α (1,2)-fucose connection is associated with the H blood group antigen usually.α (1,3)-be connected and Lewis with α (Isosorbide-5-Nitrae)-fucose XThe modification of antigen is associated.α (1,6)-fucose connects N-Acetyl-D-glucosamine (GlcNac) molecule that is connected with N-and is associated as those molecules in antibody.
The fucosylation that it is believed that protein plays a role in mammiferous growth course.The mice homozygote of FX gene target sudden change is shown multiple abnormal, comprise lethal phenotype.Also reported that mice weakened from the recovery of heterozygote cenospecies.(people such as Becker, Mammalian Genome14:130-139 (2003)).Propose abnormal protein fucosylation and be associated with human diseases, be included in the sialylated Lewis in cancer XWith sialylated Lewis yRise.These polysaccharides are parts that E-type and P-type are selected plain molecule.Sialylated Lewis on cancerous cell by inference XWith sialylated Lewis yThe raising of polysaccharide by with endothelium on E-type and P-type select the interaction of element to promote to shift.In the patient of rheumatic arthritis, also observing the fucosylation polysaccharide increases.Yet, the present treatment means for albumen fucosylation level that also there is no approved.
Summary of the invention
Methods described herein and compositions part, based on the unpredictable consequence shown in embodiment, demonstrate protein fucosylation in the animal of using the fucose analog and reduce.The fucosylation of antibody and other protein can be regulated with fucose analog as herein described.
On the one hand, the invention provides in vivo and produce and go fucosylation method of protein and compositions.Use the protein that produces fucose analog (as chemical formula I, II, III, IV, V or VI) such as cell cortex protein in animal such as mammal, the fucosylation of this animal reduces.The reduction of fucosylation is respectively with respect to without having chemical formula I, II, III, IV, the animal that the fucose analog of V or VI is processed.
In related fields, the invention provides and produce in vivo antibody that the core fucosylation reduces and the method and composition of antibody derivatives.Use the fucose analog (as chemical formula I, II, III, IV, V or VI) in animal, this animal produces antibody and the antibody derivatives (that is, the fucosylation of the N-Acetyl-D-glucosamine of the compound sugar chain of N-glucosides connecting-type reduces, and described sugar chain is incorporated into the Fc district by the N-Acetyl-D-glucosamine of sugar chain reducing end under neutral) that the core fucosylation reduces.The reduction of core fucosylation is respectively with respect to without having chemical formula I, II, III, IV, the animal that the fucose analog of V or VI is processed.
On the other hand, the invention provides and comprise the fucose analog and prepare for the pharmaceutical composition that is applied to target animal.Described fucose analog can be prepared for being applied to target animal to suppress in vivo or to reduce fucosylation.
Above-mentioned and other aspects of the present invention can be by reference to followingly describing in detail, non-limiting example and the accompanying drawing of the specific embodiment are able to comprehend.
Brief Description Of Drawings
Fig. 1 shows the impact that fucose analog (passing through lumbar injection) antagonist fucosylation is used in use.Left figure display dot trace figure and right figure displaing coordinate figure.Dot blot albumen point sample level (the picture left above) shows antibody cAC10 standard (the Dot blot leftmost side dotted line frame of below and the respective column of top Dot blot) with fucose specific biological luminous (lower-left figure), undressed contrast (Dot blot of below is the respective column of second dotted line frame and top Dot blot from left to right), and alkynyl fucose (SGD-1887
The dotted line frame rectangle that the Dot blot of below is placed in the middle and the respective column of top Dot blot), peracetic acid alkynyl fucose (SGD-1890; The Dot blot of below is the respective column of second dotted line frame and top Dot blot from right to left), and 2-fluorine fucose (SGD-2083; The dotted line frame of the Dot blot rightmost side of below and the respective column of top Dot blot).After proofreading and correct the point sample level, the percentage rate of fucosylation is presented in the coordinate diagram of right side.
Fig. 2 demonstration gives the shadow of fucose analog for antibody core fucosylation by drinking water
Ring.Coordinate diagram shows the percentage rate of the antibody fucosylation of being measured by gas chromatogram (GC): figure A and B display separation are from the fucosylation level of the anti-KLH-antibody (Ab) of processed group, and figure C and D show the level of the fucosylation of all the other IgG antibody (non--KLH-specificity).Figure A and C show the fucosylation percentage rate of each animal that the standard curve (0-100% fucosylation) of use antibody purification is definite.Figure B and the fucosylation level of D with the percentage rate display process group of relative undressed matched group average.
Fig. 3 demonstration gives the impact of fucose analog for antibody core fucosylation by drinking water.In figure, show the fucosylation level of non--KLH-specific antibody.The Dot blot that shows albumen point sample level (the picture left above) and fucose specific biological luminous (lower-left figure), be respectively the antibody cAC10 standard (Dot blot of top and below, leftmost side frame), undressed matched group (the Dot blot of top and below, second (top) and right frame from left to right), and 2-fluorine fucose (Dot blot of top and below, second (below) and second frame (top and below) from left to right from right to left).After proofreading and correct the point sample level, fucosylated percentage rate is presented in the coordinate diagram of right side.
Fig. 4 shows the impact that gives various dose 2-fluorine fucose antagonist core fucosylation by drinking water.Dot blot show albumen point sample level (left side) and fucose specific biological luminous (in), be respectively undressed matched group and 1,10, with 100 mMs of SGD-2083 (as shown in the figure).The fucosylated percentage rate of comparing with unprocessed group is presented in the coordinate diagram of right side.
Fig. 5 demonstration gives the impact of 2-fluorine fucose on circulating leukocyte and neutrophil cell.Figure A, from individual mice blood sample collection, get rid of erythrocyte with Turk solution and count definite leukocyte count on hematimeter.Figure B, be to determine the neutrophil cell number, by flow cytometry, measures the leukocyte percentage rate of Gr-1+, and for the total cellular score of (A) mensuration.Figure C, gathered the lymph node storehouse from individual mice, prepares single cell suspension, and count with hematimeter.Individual mice (the every group of n=3 of symbology; Rhombus, unprocessed; Square, 1 mM of 2-fluorine fucose (SGD-2083); Triangle, 10 mMs of 2-fluorine fucosees; Circle, 100 mMs of 2-fluorine fucosees).
Fig. 6 demonstration gives 2-fluorine fucose for the impact of E-Selectin in conjunction with neutrophil cell.Figure A, use flow cytometry to identify the example of neutrophil cell.Cell comes gate to include leukocyte alive according to forward angle light scatter and lateral angle scattering, then by result, with the rectangular histogram of describing Gr-1 dyeing, represents to identify neutrophil cell.Gate is selected positive cell, determines the percentage ratio (for the cell counting of Fig. 5 B) of positive cell, and this gate is applied in the rectangular histogram of (B).Figure B, E-Selectin is in conjunction with the example of the neutrophil cell from undressed animal (left side) and the oral animal (right side) that gives 100 mMs of 2-fluorine fucosees (SGD-2803).The rectangular histogram of Lycoperdon polymorphum Vitt show E-Selectin in conjunction with and dotted line shows the only combination of second class grade chemical.The geometrical mean in conjunction with the mensuration fluorescence intensity for E-Selectin.Figure C, to the geometric average fluorescence intensity of each zoometry E-Selectin combination in (B), and compare (every group of n=3 between group; Error line represents standard deviation).
Fig. 7 shows the impact on the protein fucosylation with some fucose analog cultured cell system.Detected cell line LS174T, PC-3, Ramos, HL-60cy and Caki-1.
Fig. 8 demonstration gives the impact of fucose analog on the mouse tumor heteroplastic transplantation model.Figure A-E shows respectively use LS174T, PC-3, Ramos, the result in the mouse tumor heteroplastic transplantation model of HL-60cy and Caki-1 cell line (through 2-fluorine fucose (SGD-2803) pretreatment).Figure F shows the result in the mouse tumor heteroplastic transplantation model that uses unprocessed LS174T cell line.
Fig. 9 shows the project (figure A) and result (figure B) of tumor vaccine model, this model carries out pre-immunity based on the A20 Mus lymphoma cell that uses deactivation, then in the situation that give or do not give fucose analog (2-fluorine fucose), uses active A 20 cells to attack.
The specific embodiment
Definition
Term " antibody " refers to the immunocompetence part of (a) immunoglobulin polypeptides and immunoglobulin polypeptides, namely, the polypeptide of immunoglobulin family, or its fragment, they comprise the antigen binding site of energy immunologic opsonin in conjunction with specific antigen, and have the Fc domain that contains compound N-glucosides connecting-type sugar chain, or (b) these immunoglobulin polypeptides polypeptide or fragment energy immunologic opsonin in conjunction with the conservative substitutive derivative of described antigen.Antibody is summarized in, for example, and Harlow and Lane, Antibodies:A Laboratory Manual (antibody: laboratory manual) (publishing house of cold spring harbor laboratory (Cold Spring Harbor Laboratory Press), 1988).
" antibody derivatives " refers to the Fc domain or the zone that comprise compound N-glucosides connecting-type sugar chain of defined antibody (comprising antibody fragment) above or antibody; usually the heterologous molecule that is not associated with antibody or Fc domain or zone by covalent bond and being modified; for example; by in conjunction with heterologous polypeptide (for example; the ligand binding domain of heterologous protein); or by glycosylation (except the core fucosylation); deglycosylation (except non-core fucosylation); acetylation; phosphorylation, or other modifications.
Term " monoclonal antibody " refers to that antibody comes from the single cell clone, comprises any eucaryon or procaryotic cell clone, or phage clone, and not refers to its production method.Therefore, term " monoclonal antibody " is not limited only to the antibody of producing by hybridoma technology.
Term " Fc district " refers to the constant region of antibody, for example, and C H1-hinge-C H2-C H3 domains, optional have a C H4 domains, or the conservative substitutive derivative in this type of Fc district.
Term " Fc domain " refers to the constant domain of antibody, for example, and C H1, hinge, C H2, C H3, or C H4 territories, or the conservative substitutive derivative of this type of Fc domain.
" antigen " is the molecule of certain antibody or antibody derivatives specific binding.
Term " specific binding " and " specific binding in " refer to that antibody or antibody derivatives will be combined its corresponding target antigen and not in conjunction with numerous other antigen in the high selectivity mode.Usually, the affinity at least 1 of antibody or antibody derivatives combination * 10 -7M, and preferred 1 * 10 -8M to 1 * 10 -9M, 1 * 10 -10M, 1 * 10 -11, or 1 * 10 -12M, and the affinity of being combined with predetermined antigens doubles it at least in conjunction with the heterogenetic antigen beyond predetermined antigens or the affinity of closely related antigen (for example, BSA, casein).
Term " inhibition " or " right ... inhibition " but refer to weaken with detection limit, or stop fully.
Unless context is otherwise noted, " alkynyl fucose peracetic acid ester " used herein refers in the R1-4 position (formula I and II vide infra) any or form of ownership with the alkynyl fucose (5-acetenyl arabinose) of aceticoceptor, comprises 6-acetenyl-tetrahydrochysene-2H-pyrans-2,3,4,5-, tetra-bases-tetracetate, comprise (2S, 3S, 4R, 5R, 6S) and (2R, 3S, 4R, 5R, 6S) isomer, and 5-((S)-1-hydroxypropyl-2-alkynyl)-oxolane-2,3,4-tri-bases-tetracetate, comprise (2S, 3S, 4R, 5R) and (2R, 3S, 4R, 5R) isomer, and the aldose form.Term " alkynyl fucose triacetate " " alkynyl fucose diacetate esters " reach " alkynyl fucose monoacetate " represent respectively three of alkynyl fucose-, two-, and list-acetas form.
Unless context is otherwise noted, term " alkyl " refers to have 1 to 20 carbon atom unsubstituted saturated straight chain or the side chain hydro carbons of (and all combinations and recombinant of carbon atom number range therebetween and concrete quantity), unless separately refer in particular to.Preferred 1 to 3, the alkyl group of 1 to 8 or 1 to 10 carbon atom.The example of alkyl group has methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl group, sec-butyl, the tert-butyl group, n-pentyl, 2-amyl group, 3-amyl group, 2-methyl-2-butyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, positive decyl, 3-methyl-2-butyl, 3-methyl isophthalic acid-butyl, 2-methyl-1-butene base, the 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-amyl group, 3-methyl-2-amyl group, 4-methyl-2-amyl group, 3-methyl-3-amyl group, 2-methyl-3-amyl group, 2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl.
No matter alkyl group, be separately or as the part of another group, when being substituted, can be replaced by one or more groups, and preferred 1 to 3 group (and any additional replacement that is selected from halogen family) includes but not limited to: halogen ,-O-(C 1-C 8Alkyl) ,-O-(C 2-C 8Thiazolinyl) ,-O-(C 2-C 8Alkynyl) ,-aryl ,-C (O) R ' ,-OC (O) R ' ,-C (O) OR ' ,-C (O) NH 2,-C (O) NHR ' ,-C (O) N (R ') 2,-NHC (O) R ' ,-SR ' ,-SO 3R ' ,-S (O) 2R ' ,-S (O) R ' ,-OH ,=O ,-NH 2,-NH (R ') ,-N (R ') 2With-CN; Wherein each R ' is independently selected from-H ,-C 1-C 8Alkyl ,-C 2-C 8Thiazolinyl ,-C 2-C 8Alkynyl or aryl.
Unless this otherwise noted, term " thiazolinyl " refers to have 2 to 20 carbon atoms with " alkynyl " the not replacement of (and all combinations and recombinant of carbon atom number range therebetween and concrete quantity) or the straight or branched carbochain of optional being substituted (have and indicate), from 2 to 3 of preferred carbon numbers, 2 to 4,2 to 8, or 2 to 10.Alkenylene chain has at least one two key in chain and the alkynyl chain has at least one triple bond in chain.The example of alkenyl group includes but not limited to, alkylidene or vinyl, pi-allyl ,-1 alkene butyl ,-2 alkene butyl ,-isobutenyl ,-1 pentenyl ,-2 pentenyls ,-3-methyl-1-butene base ,-2 methyl 2 cyclobutenyls, and-2,3 dimethyl 2 cyclobutenyls.The example of alkynyl group includes but not limited to, alkynes class, propargyl, acetenyl, propinyl ,-1 butynyl ,-2 butynyl ,-1 pentynyl ,-2 pentynyl, and-3 methyl 1 butynyl.
No matter thiazolinyl and alkynyl group, be separately or as the part of another group, when being substituted, can be replaced by one or more groups, and preferred 1 to 3 group (and any additional replacement that is selected from halogen family), include but not limited to: halogen ,-O-(C 1-C 8Alkyl) ,-O-(C 2-C 8Thiazolinyl) ,-O-(C 2-C 8Alkynyl) ,-aryl ,-C (O) R ' ,-OC (O) R ' ,-C (O) OR ' ,-C (O) NH 2,-C (O) NHR ' ,-C (O) N (R ') 2,-NHC (O) R ' ,-SR ' ,-SO 3R ' ,-S (O) 2R ' ,-S (O) R ' ,-OH ,=O ,-NH 2,-NH (R ') ,-N (R ') 2With-CN; Wherein each R ' is independently selected from-H ,-C 1-C 8Alkyl ,-C 2-C thiazolinyl ,-C 2-C 8Alkynyl or aryl.
Unless this otherwise noted, term " alkylidene " refers to have 1 to 20 carbon atom (and all combinations and recombinant of carbon atom number range therebetween and concrete quantity), preferably have 1 to 8 or 1 to 10 carbon atom, and have unsubstituted saturated side chain or straight-chain alkyl that same carbon atom by removing parent ethylene or two hydrogen atoms on two different carbon atoms obtain two monoradical centers.Typical case's alkylidene includes but not limited to, methylene, and ethylidene, propylidene, butylidene, pentylidene, hexylidene, inferior heptyl, octylene, nonamethylene, inferior decyl, Isosorbide-5-Nitrae-cyclohexylene etc.
No matter alkylidene group, be separately or as the part of another group, when being substituted, can be replaced by one or more groups, and preferred 1 to 3 group (and any additional replacement that is selected from halogen), include but not limited to: halogen ,-O-(C 1-C 8Alkyl) ,-O-(C 2-C 8Thiazolinyl) ,-O-(C 2-C 8Alkynyl) ,-aryl ,-C (O) R ' ,-OC (O) R ' ,-C (O) OR ' ,-C (O) NH 2,-C (O) NHR ' ,-C (O) N (R ') 2,-NHC (O) R ' ,-SR ' ,-SO 3R ' ,-S (O) 2R ' ,-S (O) R ' ,-OH ,=O ,-NH 2,-NH (R ') ,-N (R ') 2With-CN; Wherein each R ' is independently selected from H ,-C 1-C 8Alkyl ,-C 2-C 8Thiazolinyl ,-C 2-C 8Alkynyl or-aryl.
Term " alkenylene " refers to the undersaturated side chain of thiazolinyl (as mentioned above) or the hydro carbons group of straight chain or ring-type, and has the group center that same carbon atom by removing parent alkene or two hydrogen atoms on two different carbon atoms obtain two unit prices.For as described in thiazolinyl, " alkenylene " group can refer to unsubstituted or optional substituted (indicating) as above.In some embodiments, " alkenylene " group is not substituted.
Term " alkynylene " refers to the undersaturated side chain of alkynyl (as mentioned above) or the hydro carbons group of straight chain or ring-type, and two hydrogen atoms with same carbon atom by removing parent alkynes or two different carbon atoms obtain the group center of two unit prices.For as described in alkynyl, " alkynylene " group can refer to unsubstituted or optional substituted (indicating) as above.In some embodiments, " alkynylene " group is not substituted.
Unless this otherwise noted, term " aryl " refers to the substituted of 6 to 20 carbon atoms (and all combinations and recombinant of carbon atom number range therebetween and concrete quantity) or unsubstituted monovalent aromatic family hydrocarbyl group, by a hydrogen atom on the single carbon atom of removing parent aromatic ring system, obtains.Some aromatic yl groups are expressed as " Ar " in example arrangement.Typical case's aromatic yl group includes but not limited to, is derived from benzene, substituted benzene, phenyl, naphthalene, anthracene, biphenyl etc.
No matter aromatic yl group, be separately or as the part of another group, when being substituted, can be replaced by one or more groups, and preferred 1 to 5 group, even 1 to 2 group, include but not limited to: halogen ,-C 1-C 8Alkyl ,-C 2-C 8Thiazolinyl ,-C 2-C 8Alkynyl ,-O-(C 1-C 8Alkyl) ,-O-(C 2-C 8Thiazolinyl) ,-O-(C 2-C 8Alkynyl) ,-aryl ,-C (O) R ' ,-OC (O) R ' ,-C (O) OR ' ,-C (O) NH 2,-C (O) NHR ' ,-C (O) N (R ') 2,-NHC (O) R ' ,-SR ' ,-SO 3R ' ,-S (O) 2R ' ,-S (O) R ' ,-OH ,-NO 2,-NH 2,-NH (R ') ,-N (R ') 2With-CN; Wherein each R ' is independently selected from H ,-C 1-C 8Alkyl ,-C 2-C 8Thiazolinyl ,-C 2-C 8Alkynyl or aryl.
Unless this otherwise noted, term " heterocycle " refers to have from 3 to 7, or the loop systems of the substituted or unsubstituted monocycle of 3 to 10 annular atomses (also referred to as ring members), wherein at least one annular atoms is to be selected from N, O, the hetero atom of P or S (and all combinations and recombinant of carbon atom therebetween and heteroatomic scope and concrete quantity).Described heterozygosis can have and independently is selected from N, O, the 1-4 of a P or S hetero atom.One or more N in heterocycle, O or S atom can be oxidized.The heterocycle of monocycle preferably has 3-7 ring members (for example, 2-6 carbon atom and independently be selected from N, O, the 1-3 of a P or S hetero atom).Comprising heteroatomic ring can be aromatic ring or non-aromatic ring.Unless otherwise noted, heterocycle connects its side group on any hetero atom that can produce rock-steady structure or carbon atom.
Paquette " Principles of Modern Heterocyclic Chemistry " (contemporary heterocyclic chemistry principle) (W.A.Benjamin, New York, 1968) are shown in the description of heterocycle, particularly the 1st, 3,4,6,
7 and 9 chapters; " The Chemistry of Heterocyclic Compounds, A series ofMonographs " (chemistry of heterocyclic compound book series) ((John Wiley&amp of New York John Wei Lisen publishing house; Sons), 1950 so far), the 13rd, 14,16,19 and 28 volumes particularly; And J.Am.Chem.Soc.82:5566 (1960).The example of " heterocycle " group includes but not limited to pyridine radicals, dihydropyridine base, tetrahydro pyridyl (piperidyl), thiazolyl, pyrimidine radicals, furyl, thienyl, pyrrole radicals, pyrazolyl, imidazole radicals, tetrazole radical, fucosido, '-aziridino (azirdinyl), azelidinyl (azetidinyl), epoxy ethyl, glycidyl and tetrahydrofuran base.
No matter heterocyclic group, be separately or as the part of another group, when being substituted, can be replaced by one or more groups, and preferred 1 to 2 group, include but not limited to :-C 1-C 8Alkyl ,-C 2-C 8Thiazolinyl ,-C 2-C 8Alkynyl, halogen ,-O-(C 1-C 8Alkyl) ,-O-(C 2-C 8Thiazolinyl) ,-O-(C 2-C 8Alkynyl) ,-aryl ,-C (O) R ' ,-OC (O) R ' ,-C (O) OR ' ,-C (O) NH 2,-C (O) NHR ' ,-C (O) N (R ') 2,-NHC (O) R ' ,-SR ' ,-SO 3R ' ,-S (O) 2R ' ,-S (O) R ' ,-OH ,-NH 2,-NH (R ') ,-N (R ') 2With-CN; Wherein each R ' is independently selected from H ,-C 1-C 8Alkyl ,-C 2-C 8Thiazolinyl ,-C 2-C 8Alkynyl or-aryl.
Exemplary but nonrestrictive, the carbon bond heterocycle can be with upper/lower positions Cheng Jian: 2,3,4,5 or No. 6 positions of pyridine; 3,4,5 or No. 6 positions of pyridazine; 2,4,5 or No. 6 positions of pyrimidine; 2,3,5 or No. 6 positions of pyrazine; 2,3,4 or No. 5 positions of furan, oxolane, thio-furan, thiophene, pyrroles or nafoxidine;
Figure BDA00002816630300081
2,4 or No. 5 positions of azoles, imidazoles, thiazole; Different
Figure BDA00002816630300082
3,4 or No. 5 positions of azoles, pyrazoles or isothiazole; 2 or No. 3 positions of aziridine; Or 2,3 or No. 4 positions of azetidine.Exemplary bond with carbon heterocycle can comprise the 2-pyridine radicals, 3-pyridine radicals, 4-pyridine radicals, 5-pyridine radicals, the 6-pyridine radicals, 3-pyridazinyl, 4-pyridazinyl, the 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidine radicals, the 4-pyrimidine radicals, 5-pyrimidine radicals, 6-pyrimidine radicals, the 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl or 5-thiazolyl.
Exemplary but nonrestrictive, nitrogen key heterocycle can be with upper/lower positions Cheng Jian: ethylene imide, azepine
Tetramethylene., pyrroles, Pyrrolizidine, 2-pyrrolin, 3-pyrrolin, imidazoles, imidazolidine, 2-imidazoline,
No. 1 position of 3-imidazoline, pyrazoles, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidines, piperazine, indole, indoline or 1 hydrogen-indazole; No. 2 positions of iso-indoles or isoindoline; No. 4 positions with morpholine
。More typically, nitrogen key heterocycle comprises 1-ethylene imide base, 1-azelidinyl, 1-pyrrole radicals, 1-imidazole radicals, 1-pyrazolyl, and piperidino.
Unless this otherwise noted, term " carbocyclic ring " refers to have 3 to 6 annular atomses (and all combinations and recombinant of carbon atom number range therebetween and concrete quantity) substituted or unsubstituted, the loop systems of saturated or unsaturated non-aromatic monocycle, wherein all annular atomses are all carbon atoms.
No matter carbon ring group is separately or as the part of another group, when being substituted, for example can be by following one or more groups, preferred 1 or 2 group (and any additional substituent group that is selected from halogen) replaces, and includes but not limited to: halogen ,-C 1-C 8Alkyl ,-C 2-C 8Thiazolinyl ,-C 2-C 8Alkynyl ,-O-(C 1-C 8Alkyl) ,-O-(C 2-C 8Thiazolinyl) ,-O-(C 2-C 8Alkynyl), aryl ,-C (O) R ' ,-OC (O) R ' ,-C (O) OR ' ,-C (O) NH 2,-C (O) NHR ' ,-C (O) N (R ') 2,-NHC (O) R ' ,-SR ' ,-SO 3R ' ,-S (O) 2R ' ,-S (O) R ' ,-OH ,=O ,-NH 2,-NH (R ') ,-N (R ') 2With-CN; Wherein each R ' is independently selected from-H ,-C 1-C 8Alkyl ,-C 2-C 8Thiazolinyl ,-C 2-C 8Alkynyl or aryl.
The substituent example of the carbocyclic ring of monocycle comprises cyclopropyl, cyclobutyl, cyclopenta, 1-ring penta-1-thiazolinyl, 1-ring penta-2-thiazolinyl, 1-ring penta-3-thiazolinyl, cyclohexyl, 1-hexamethylene-1-thiazolinyl, 1-hexamethylene-2-thiazolinyl, 1-hexamethylene-3-thiazolinyl, suberyl, ring octyl group,-1,3-cyclohexyl dialkylene ,-Isosorbide-5-Nitrae-cyclohexyl dialkylene,-1,3-suberyl dialkylene ,-1,3,5-suberyl trialkenyl, and-ring octyl group dialkylene.
In any ingredient or any molecular formula, repeatedly the time, the definition when it occurs at every turn is separate when any occurrences.The combination of substituent group and/or variable only is only permissible when this combination results stable compound.
Unless this otherwise noted, hyphen (-) is indicated the junction point with the side group molecule.Therefore, term " (C 1-C 10Alkylidene) aryl " or " C 1-C 10Alkylidene (aryl) " refer to C as defined herein 1-C 10Alkylidene group, wherein said alkylidene group connects side group molecule by its any one carbon atom and is replaced by aryl as herein defined with the hydrogen atom of carbon atom Cheng Jian in described alkylidene group.
When special groups " was substituted ", described group can have the one or more substituent groups that independently are selected from the substituent group list, preferred one to five substituent group, more preferably one to three substituent group, most preferably one to two substituent group.Yet described group can have the substituent group of any amount that is selected from halogen usually.
According to inventor intention, to any substituent group of the ad-hoc location of molecule or the definition of variable, should be independent of its definition in this other position of molecule.Should be understood that those skilled in the art can select substituent group and the replacement mode on compound of the present invention, to provide activity and chemically stable and to be easy to techniques known in the art and the synthetic compound of methods described herein.
Term " pharmaceutically acceptable " refers to ratify or list in American Pharmacopeia or other putative pharmacopeia through federal government or regulator of state government, for animal, or specifically for the people's.Term " pharmacy compatible ingredients " refers to pharmaceutically acceptable diluent, adjuvant, the excipient that fucose can together give with it, or supporting agent.
" little electron withdraw group " refer at substituent group connection site place, and the substituent group of comparing for example hydrogen atom or hydroxyl or relatively being arranged in this site of fucose has stronger electronegative any substituent group.Usually, described little electron withdraw group has 10 or atom still less (except hydrogen atom) and comprises group such as nitro; Cyano group and cyanoalkyl (for example ,-CH 2CH 2CN); Halogen; Acetylene or other alkynes or halo alkynes ((C ≡ CCF for example 3); Alkene or alkenyl halide; The allene class; Carboxylate, esters, amide-type and halo form thereof; Sulfonate and phosphonate or ester, esters, amide-type and halo form thereof; Halogenated alkyl group (for example ,-CF 3,-CHF 2,-CH 2CF 3), acyl group and halogenacyl group (for example ,-C (O) CH 3With-C (O) CF 3); Alkyl sulphonyl and halogenated alkyl sulfonyl (for example ,-S (O) 2Alkyl and-S (O) 2Haloalkyl); Aryloxy group (for example, phenoxy group and substituted phenoxy group); Aralkoxy (for example, benzyloxy and substituted benzyloxy); And oxirane.Little electron withdraw group preferably has 8,7,6 or atom still less (except hydrogen atom).
The fucose analog does not contain in fact unwanted pollutant usually.This means that the common purity of described analog is at least about 50%w/w (w/w), also do not contain in fact simultaneously and disturb albumen and other pollutant.Reagent purity is sometimes at least about 80%w/w, more preferably 90%w/w or about 95%w/w at least.Use conventional purification technique, can be obtained up to the homogenizing product of few 99%w/w.
General introduction
The invention provides the method and the compositions that in animal individual, reduce the protein fucosylation.The proposition of described method partly is based on the afterclap that shows in embodiment, namely, by the fucose analog, give the core fucosylation reduction that object (for example, mammal) causes antibody or antibody derivatives, and the fucosylation of other albumen also reduces." fucosylation reduction " with regard to protein be often referred to reduce on polysaccharide by α (1,2)-, α (1,3)-, α (Isosorbide-5-Nitrae)-, the fucose addition of α (1,6)-connection." core fucosylation " with regard to antibody refers to fucose addition (" fucosylation ") to the N-Acetyl-D-glucosamine (" GlcNAc ") at the reducing end under neutral place of the N-connecting-type polysaccharide of antibody.The fucose that " reduction of core fucosylation " with regard to antibody refers to be connected on the reducing end under neutral place N-Acetyl-D-glucosamine (" GlcNAc ") of N-connecting-type polysaccharide of antibody reduces than undressed animal.
In many aspects as herein described, accept normally mammal of animal that the fucose analog gives, be preferably the mankind.Therefore, the present invention also is provided for reducing the method and composition of the protein fucosylation in mammal such as the mankind.
In other respects, provide the pharmaceutical composition that is suitable for giving animal of fucose analog and pharmaceutical excipient, wherein by the fucose analog of effective dose and described mixed with excipients together.In some embodiments, described fucose analog is dried forms (for example, lyophilizing), alternatively with the stabilizing agent of the stability that can improve described compositions for long-term storage.In some embodiments, the pharmaceutical composition of fucose analog and pharmaceutical excipient is mixed with for animals administer.In some further embodiments, the pharmaceutical composition of fucose analog and pharmaceutical excipient is mixed with the administration for the mankind.
In some embodiments, the fucosylation of compound N-glucosides connecting-type sugar chain that is incorporated into the Fc district (or domain) of antibody is lowered.As used herein, " compound N-glucosides connecting-type sugar chain " is incorporated into agedoite 297 (pressing Kabat numbering system numbering) combination usually, although compound N-glucosides connecting-type sugar chain also can be connected on other asparagine residue.As described herein, compound N-glucosides connecting-type sugar chain has the compound sugar chain of two feeler types, mainly has following structure:
Figure BDA00002816630300121
Wherein ± refer to can contain also and can lack this glycan molecule, and digital watch understands the position of the connection between glycan molecule.In said structure, the end that sugar chain is combined with agedoite is called reducing end under neutral (right side), and opposite side is called non reducing end.Fucose is incorporated into the N-Acetyl-D-glucosamine (" GlcNAc ") of reducing end under neutral usually, usually by α 1,6 key (the 6-position of GlcNAc is in conjunction with the 1-position of fucose)." Gal " refers to galactose, and " Man " refers to mannose.
The high mannose type sugar chain of mannose is only included in " compound N-glucosides connecting-type sugar chain " eliminating at the non reducing end place of core texture, but comprise 1) complex-type sugar chain, non reducing end one side of its core texture have one or more galactose-N-Acetyl-D-glucosamine (also referred to as " and gal-GlcNAc) branch, and non reducing end one side of Gal-GlcNAc optionally has sialic acid, sections type N-Acetyl-D-glucosamine or similar structures; Perhaps 2) heterozygous, non reducing end one side of its core texture has high mannose N-glucosides connecting-type sugar chain and compound N-glucosides connecting-type sugar chain simultaneously.
In some embodiments, " compound N-glucosides connecting-type sugar chain " comprises compound, the non reducing end of its core texture has zero, one or more galactose-N-Acetyl-D-glucosamine (also referred to as " gal-GlcNAc) branch and non reducing end one side of Gal-GlcNAc optionally also have sialic acid, section type N-Acetyl-D-glucosamine or similar structures, but get rid of the sugar chain with high mannose composition.
The method according to this invention, after giving the fucose analog, only have a small amount of fucose can be included into sugar chain (for example, polysaccharide or compound N-glucosides connecting-type sugar chain) usually.For example, in different embodiments, with the animal of not accepting the fucose analog, compare, described animal (for example, mammal such as the mankind) in serum, lower than approximately 60%, lower than approximately 50%, lower than approximately 40%, lower than approximately 30%, lower than approximately 20%, lower than approximately 15%, lower than approximately 10%, lower than approximately 5% or lower than about 1% antibody by the core fucosylation.In some embodiments, with the animal of processing without the fucose analog, compare, the antibody in animal serum is not in fact by (for example, lower than 0.5%) core fucosylation.
In some embodiments, with the animal of not accepting the fucose analog, compare, animal (for example, mammal such as people) in regard to the fucosylation cell cortex protein, the protein fucosylation reduces, degree approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 15%, approximately 10%, approximately 5% or approximately 1%.In some embodiments, with the animal of not accepting the fucose analog, compare, animal (for example, mammal such as people) in regard to the fucosylation cell surface protein, by α (1,2)-protein fucosylation of connecting reduces, degree approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 15%, approximately 10%, approximately 5% or approximately 1%.
In some embodiments, with the animal of not accepting the fucose analog, compare, animal (for example, mammal such as people) in regard to the fucosylation cell surface protein, by α (1,3)-protein fucosylation of connecting reduces, degree approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 15%, approximately 10%, approximately 5% or approximately 1%.In some embodiments, with the animal of not accepting the fucose analog, compare, animal (for example, mammal such as people) in regard to the cell surface protein of fucosylation, by α (1,4)-protein fucosylation of connecting reduces, degree approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 15%, approximately 10%, approximately 5% or approximately 1%.
In some embodiments, with the animal of not accepting the fucose analog, compare, animal (for example, mammal such as people) in regard to the cell surface protein of fucosylation, by α (1,6)-protein fucosylation of connecting reduces, degree approximately 60%, approximately 50%, approximately 40%, approximately 30%, approximately 20%, approximately 15%, approximately 10%, approximately 5% or approximately 1%.
In some embodiments, with the animal of not accepting the fucose analog, compare, animal (for example, mammal such as people) leukocytic fucosylation reduces in serum, and degree is at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 15%, at least about 10% or at least about 5%.In some embodiments, with the animal of not accepting the fucose analog, compare, animal (for example, mammal such as people) in serum leukocyte by α (1,3)-fucosylation of connecting reduces, and degree is at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 15%, at least about 10% or at least about 5%.In some embodiments, with the animal of not accepting the fucose analog, compare, animal (for example, mammal such as people) in serum leukocyte by α (1,4)-fucosylation of connecting reduces, and degree is at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 15%, at least about 10% or at least about 5%.
In some embodiments, with the animal of not accepting the fucose analog, compare, animal (for example, mammal such as people) fucosylation of Serum Antibody reduces, and degree is at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 15%, at least about 10% or at least about 5%.
In some embodiments, only have a small amount of fucose analog (or the metabolite of described fucose analog or product) to be included into the polysaccharide (for example described compound N-glucosides connecting-type sugar chain) of described antibody, antibody derivatives or the polysaccharide of other protein.For example, in different embodiments, with the animal of not accepting the fucose analog, compare, lower than approximately 60%, lower than approximately 40%, lower than approximately 30%, lower than approximately 20%, lower than approximately 15%, lower than approximately 10%, lower than approximately 5% or be included on the polysaccharide of antibody in described animal serum lower than about 1% fucose analog (or the metabolite of described fucose analog or product).In some embodiments, with the animal of not accepting the fucose analog, compare, lower than approximately 60%, lower than approximately 40%, lower than approximately 30%, lower than approximately 20%, lower than approximately 15%, lower than approximately 10%, lower than approximately 5% or lower than about 1% fucose analog (or the metabolite of described fucose analog or product), be included on the polysaccharide of cell surface protein of animal.
In some embodiments, with the animal of not accepting the fucose analog, compare, lower than approximately 60%, lower than approximately 40%, lower than approximately 30%, lower than approximately 20%, lower than approximately 15%, lower than approximately 10%, lower than approximately 5% or be included in animal serum on leukocytic polysaccharide lower than about 1% fucose analog (or the metabolite of described fucose analog or product).
The fucose analog
The suitable fucose analog of the method for the invention (hereinafter being decided to be respectively chemical formula I, II, III, IV, V and VI) is to give safely with effective dose those fucose analog of the core fucosylation of the compound N-glucosides connecting-type sugar chain that animal suppresses antibody or antibody derivatives.The fucose analog has description in published U.S. Patent application 2009-0317869, the fucose of the antibody that it produces at external reduction host cell or the compound N-glucosides connecting-type sugar chain of antibody derivatives is included in.The fucose analog can be outer by gastrointestinal tract, oral or other suitable administering mode give animal (for example, mammal).
In some embodiments, fucose analog (or born of the same parents' intracellular metabolite thing or product of described fucose analog) suppresses the enzyme of fucose in remedying.(as described herein, born of the same parents' intracellular metabolite thing can be the analog of for example GDP-modification or the analog that takes off wholly or in part ester.Product can be for example to take off wholly or in part the analog of ester.) for example, fucose analog (or born of the same parents' intracellular metabolite thing or product of described fucose analog) can suppress the activity of Fucokinase or GDP-fucose-pyrophosphorylase.In some embodiments, fucose analog (or born of the same parents' intracellular metabolite thing or product of described fucose analog) (for example suppresses fucosyltransferase, 1,2-fucosyltransferase, 1, the 3-fucosyltransferase, Isosorbide-5-Nitrae-fucosyltransferase, or 1,6-fucosyltransferase (for example, FUT8 albumen)).In some embodiments, fucose analog (or born of the same parents' intracellular metabolite thing or product of described fucose analog) can suppress the activity of the enzyme in fucose de novo synthesis path.For example, fucose analog (or born of the same parents' intracellular metabolite thing or product of described fucose analog) can suppress GDP-mannose 4, the activity of 6-dehydratase or/or GDP-fucose synzyme.In certain embodiments, fucose analog (or born of the same parents' intracellular metabolite thing or product of described fucose analog) can suppress fucose transport protein (for example, GDP-fucose transport protein).
In some embodiments, described fucose analog has following chemical formula I or (II):
Perhaps biologically acceptable salt or the solvate of described analog, wherein chemical formula I or (II) can be α or β anomer or corresponding aldose form separately.In above-mentioned chemical formula, R1 to R 4Be selected from independently of one another lower group :-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle) ,-OC (O) CH 2O (CH 2CH 2O) nCH 3,-OC (O) CH 2CH 2O (CH 2CH 2O) nCH 3,-O-tri--C 1-C 3The alkyl silicyl ,-OC 1-C 10Alkyl ,-OCH 2OC (O) alkyl ,-OCH 2OC (O) thiazolinyl ,-OCH 2OC (O) alkynyl ,-OCH 2OC (O) aryl ,-OCH 2OC (O) heterocycle ,-OCH 2OC (O) O alkyl ,-OCH 2OC (O) O thiazolinyl ,-OCH 2OC (O) O alkynyl ,-OCH 2OC (O) O aryl ,-OCH 2OC (O) O heterocycle, wherein each n is the integer that independently is selected from 0-5; And R 5Be selected from lower group :-C ≡ CH ,-C ≡ CCH 3,-CH 2C ≡ CH ,-C (O) OCH 3,-CH (OAc) CH 3,-CN ,-CH 2CN ,-CH 2X (wherein X is F, Br, Cl or I) ,-CHX 2(wherein each X is F, Br or Cl) and methyl oxirane (methoxiran).
In some embodiments, described fucose analog has chemical formula I or (II), wherein, and R 1To R 4Be selected from independently of one another lower group :-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) CH 2O (CH 2CH 2O) nCH 3,-OC (O) CH 2CH 2O (CH 2CH 2O) nCH 3,-O-tri--C 1-C 3Silicyl ,-OC 1-C 10Alkyl ,-OCH 2OC (O) alkyl ,-OCH 2OC (O) O alkyl ,-OCH 2OC (O) aryl, and-OCH 2OC (O) O aryl, wherein each n is the integer that independently is selected from 0-5; And R 5Be selected from lower group :-C ≡ CH ,-C ≡ CCH 3,-CH 2C ≡ CH ,-C (O) OCH 3,-CH (OAc) CH 3,-CN ,-CH 2CN ,-CH 2X (wherein X is F, Br, Cl or I) ,-CHX 2(wherein each X is F, Br or Cl) and methyl oxirane.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another lower group :-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle); And R 5Be selected from lower group :-C ≡ CH ,-C ≡ CCH 3,-CH 2C ≡ CH ,-C (O) OCH 3,-CH (OAc) CH 3,-CN ,-CH 2CN ,-CH 2X (wherein X is F, Br, Cl or I) ,-CHX 2(wherein each X is F, Br or Cl) and methyl oxirane.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another lower group :-O-tri--C 1-C 3Silicyl ,-OC 1-C 10Alkyl; And R 5Be selected from lower group :-C ≡ CH ,-C ≡ CCH 3,-CH 2C ≡ CH ,-C (O) OCH 3,-CH (OAc) CH 3,-CN ,-CH 2CN ,-CH 2X (wherein X is Br, Cl or I), and methyl oxirane.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another lower group :-OCH 2OC (O) alkyl ,-OCH 2OC (O) thiazolinyl ,-OCH 2OC (O) alkynyl ,-OCH 2OC (O) aryl ,-OCH 2OC (O) heterocycle ,-OCH 2OC (O) O alkyl ,-OCH 2OC (O) O thiazolinyl ,-OCH 2OC (O) O alkynyl ,-OCH 2OC (O) O aryl ,-OCH 2OC (O) O heterocycle; And R 5Be selected from lower group :-C ≡ CH ,-C ≡ CCH 3,-CH 2C ≡ CH ,-C (O) OCH 3,-CH (OAc) CH 3,-CN ,-CH 2CN ,-CH 2X (wherein X is F, Br, Cl or I) ,-CHX 2(wherein each X is F, Br or Cl) and methyl oxirane.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another lower group :-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle); And R 5Be selected from lower group :-C ≡ CH ,-C ≡ CCH 3,-CH 2C ≡ CH ,-C (O) OCH 3,-CH (OAc) CH 3,-CN ,-CH 2CN, and methyl oxirane.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another lower group :-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle); And R 5Be selected from lower group :-CH 2F ,-CH 2I ,-CH 2Br, and-CH 2Cl.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another lower group :-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle); And R 5Be selected from lower group :-CHF 2,-CHBr 2, and-CHCl 2.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another lower group :-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle); And R 5Be selected from lower group :-C ≡ CH ,-C ≡ CCH 3, and-CH 2C ≡ CH.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another lower group :-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle); And R 5Be selected from lower group-C ≡ CH ,-C ≡ CCH 3,-(CH 2) n(CN) (wherein n=0 or 1) and-C (O) OCH 3.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another lower group :-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle); And R 5Be selected from lower group :-C ≡ CH ,-C ≡ CCH 3,-CH 2CN and-C (O) OCH 3.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another lower group :-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle); And R 5Be selected from lower group :-C ≡ CH ,-C ≡ CCH 3,-CH (OAc) CH 3,-CH 2CN, and-C (O) OCH 3.
In more different embodiment, described fucose analog has chemical formula I or (II), wherein R 5As defined herein, and R 1To R 4Respectively do for oneself hydroxyl or-OC (O) C 1-C 10Alkyl.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 5As defined herein, and R 1To R 4Respectively do for oneself hydroxyl or-OAc.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another :-OH and-OC (O) C 1-C 10Alkyl; And R 5Be selected from lower group :-C ≡ CH ,-C ≡ CCH 3,-CH (OAc) CH 3,-CH 2CN ,-C (O) OCH 3,-CH 2F and-CHF 2.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another :-OH and-OAc; And R 5Be selected from lower group :-C ≡ CH ,-C ≡ CCH 3,-CH (OAc) CH 3,-CH 2CN ,-C (O) OCH 3,-CH 2F and-CHF 2.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another :-OH and-OC (O) C 1-C 10Alkyl; And R 5Be selected from lower group :-C ≡ CH ,-CH 2F and-CHF 2.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another :-OH and-OAc; And R 5Be selected from lower group :-C ≡ CH ,-CH 2F and-CHF 2.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another :-OH and-OC (O) C 1-C 10Alkyl; And R 5For-C ≡ CH.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another :-OH and-OAc; And R 5For-C ≡ CH.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another :-OH and-OC (O) C 1-C 10Alkyl; And R 5For-CHF 2.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another :-OH and-OAc; And R 5For-CHF 2.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Respectively do for oneself-OH or be selected from the ester of lower group, comprising :-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle) ,-OC (O) CH 2O (CH 2CH 2O) nCH 3(wherein n is 0-5) ,-OC (O) CH 2CH 2O (CH 2CH 2O) nCH 3(wherein n is 0-5); And R 5Be selected from lower group :-C ≡ CH ,-C ≡ CCH 3,-CH 2C ≡ CH ,-C (O) OCH 3,-CH (OAc) CH 3,-CN ,-CH 2CN ,-CH 2X (wherein X is F, Br, Cl or I) ,-CHX 2(wherein each X is F, Br or Cl) and methyl oxirane.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another :-OH and-OC (O) C 1-C 10Alkyl; And R 5For-CH 2X (wherein X is F, Br, Cl or I).
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another :-OH and-OAc; And R 5For-CH 2X (wherein X is F, Br, Cl or I).
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another :-OH and-OC (O) C 1-C 10Alkyl; And R 5For-CH 2Br.
In some embodiments, described fucose analog has chemical formula I or (II), wherein R 1To R 4Be selected from independently of one another :-OH and-OAc; And R 5For-CH 2Br.
In some embodiments, the molecular weight of described fucose analog is lower than 2000 dalton.In some embodiments, the molecular weight of described fucose analog is lower than 1000 dalton.
In some embodiments, R 5Be not substituted.
In some embodiments, each R 1To R 4Be not substituted.
In some embodiments, R 5Not ketone (C (O) alkyl).
In some embodiments, R 5Be not-CH (CH 3) OAc.
In some embodiments, as each R 1To R 4Be-during OAc, R 5Be not-CH (CH 3) OAc.
In some embodiments, R 5Be not-C ≡ CH.
In some embodiments, as any R 1To R 4Be-when OAc, R 5Be not-C ≡ CH.
In some embodiments, as any R 1To R 4When being-OC (O) alkyl, R 5Be not-C ≡ CH.
In some embodiments, each R 1To R 4When being-OC (O) alkyl, R 5Be not-C ≡ CH.
In some embodiments, as each R 1To R 4Be-when OH, R 5Be not-C ≡ CH.
In some embodiments, described fucose analog is alkynyl fucose peracetic acid ester.In some embodiments, described fucose analog is alkynyl fucose triacetate.In some embodiments, described fucose analog is alkynyl fucose diacetate esters.In some embodiments, described fucose analog is alkynyl fucose peracetic acid ester, the mixture of alkynyl fucose triacetate and alkynyl fucose diacetate esters.
In some embodiments, described fucose analog is alkynyl fucose peracetic acid ester, alkynyl fucose triacetate, the mixture of alkynyl fucose diacetate esters and alkynyl fucose monoacetate.
In some embodiments, described fucose analog is not fucose.In some embodiments, described fucose analog is not alkynyl fucose peracetic acid ester.In some embodiments, the fucose analog is not galactose or L-galactose.
In another group embodiment, described fucose analog has following chemical formula III or (IV):
Figure BDA00002816630300201
Perhaps its biologically acceptable salt or solvate, wherein each chemical formula III or (IV) they can be α or β anomer or corresponding aldose form; And in formula,
R 1To R 4Be selected from independently of one another lower group: fluorine, chlorine ,-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle) ,-OCH 2OC (O) alkyl ,-OCH 2OC (O) O alkyl ,-OCH 2OC (O) aryl ,-OCH 2OC (O) O aryl ,-OC (O) CH 2O (CH 2CH 2O) nCH 3,-OC (O) CH 2CH 2O (CH 2CH 2O) nCH 3,-O-tri--C 1-C 3Alkyl silicyl and-OC 1-C 10Alkyl, wherein each n is the integer that independently is selected from 0-5; And
Each R 2aTo R 3aBe independently selected from lower group: H, F and Cl;
R 5Be selected from lower group :-CH 3,-CHF 2,-CH=C=CH 2,-C ≡ CH ,-C ≡ CCH 3,-CH 2C ≡ CH ,-C (O) OCH 3,-CH (OAc) CH 3,-CN ,-CH 2CN ,-CH 2X (wherein X is F, Br, Cl or I), and methyl oxirane;
Wherein, R 5Be not-CH=C=CH 2,-CH 2F ,-CHF 2The time, R 1, R 2, R 3, R 2aWith R 3aWherein at least one is fluorine or chlorine.
In some embodiments of chemical formula III or (IV), R 1For F.
In some embodiments of chemical formula III or (IV), R 2For F.
In some embodiments of chemical formula III or (IV), R 3For F.
In some embodiments of chemical formula III or (IV), R 1With R 2Be respectively F.
In some embodiments of chemical formula III or (IV), R 2With R 2aBe respectively F.
In some embodiments of chemical formula III or (IV), R 1, R 3With R 4Be selected from independently of one another :-OH and-OC (O) C 1-C 10Alkyl; R 2For F; And R 5For-CH 3.
In some embodiments of chemical formula III or (IV), R 1, R 3With R 4Be selected from independently of one another :-OH and-OAc; R 2For F; And R 5For-CH 3.
In some embodiments of chemical formula III or (IV), R 1, R 3With R 4Be selected from independently of one another :-OH and-OC (O) C 1-C 10Alkyl; R 2For F; R 2aWith R 3aBe respectively H; And R 5For-CH 3.
In some embodiments of chemical formula III or (IV), R 1, R 3With R 4Be selected from independently of one another :-OH and-OAc; R 2For F; R 2aWith R 3aBe respectively H; And R 5For-CH 3.
In some embodiments of chemical formula III or (IV), R 1, R 2, R 3With R 4Be selected from independently of one another :-OH and-OC (O) C 1-C 10Alkyl; R 2aWith R 3aBe respectively H; And R 5For-CHF 2.
In some embodiments of chemical formula III or (IV), R 1, R 2, R 3With R 4Be selected from independently of one another :-OH and-OAc; R 2aWith R 3aBe respectively H; And R 5For-CHF 2.
In some embodiments of chemical formula III or (IV), R 1, R 2, R 3With R 4Be selected from independently of one another :-OH and-OC (O) C 1-C 10Alkyl; R 2aWith R 3aBe respectively H; And R 5For-CH 2F.
In some embodiments of chemical formula III or (IV), R 1, R 2, R 3With R 4Be selected from independently of one another :-OH and-OAc; R 2aWith R 3aBe respectively H; And R 5For-CH 2F.
In another group embodiment, described fucose analog has following chemical formula (V) or (VI):
Figure BDA00002816630300221
Or its biologically acceptable salt or solvate, wherein each chemical formula (V) or (VI) they can be α or β anomer or corresponding aldose form; And in formula,
Each R 1, R 2, R 2a, R 3, R 3a, R4 is selected from lower group independently of one another :-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle) ,-OCH 2OC (O) alkyl ,-OCH 2OC (O) O alkyl ,-OCH 2OC (O) aryl ,-OCH 2OC (O) O aryl ,-OC (O) CH 2O (CH 2CH 2O) nCH 3,-OC (O) CH 2CH 2O (CH 2CH 2O) nCH 3,-O-tri--C 1-C 3The alkyl silicyl ,-OC 1-C 10Alkyl, and little electron withdraw group, wherein each n is the integer that is independently selected from 0-5;
R 5Be selected from lower group :-CH 3,-CHX 2-CH 2X, that replaced by halogen or unsubstituted-CH (X ')-C 1-C 4Alkyl, that replaced by halogen or unsubstituted-CH (X ')-C 2-C 4Thiazolinyl, that replaced by halogen or unsubstituted-CH (X ')-C 2-C 4Alkynyl ,-CH=C (R 10) (R 11) ,-C (CH 3)=C (R 12) (R 13) ,-C (R 14)=C=C (R 15) (R 16), replace or unsubstituted-C by methyl or halogen 3Carbocyclic ring, by methyl or halogen, replaced or unsubstituted-CH (X ')-C 3Carbocyclic ring, replace or the unsubstituted C by methyl or halogen 3Heterocycle, by methyl or halogen, replaced or unsubstituted-CH (X ')-C 3Heterocycle ,-CH 2N 3,-CH 2CH 2N 3, and benzyloxymethyl, or R 5It is a little electron withdraw group; R wherein 10Hydrogen or the C that is not substituted or replaced by halogen 1-C 3Alkyl; R 11The C that is not substituted or is replaced by halogen 1-C 3Alkyl; R 12Hydrogen, halogen or the C that is not substituted or is replaced by halogen 1-C 3Alkyl; R 13Hydrogen, or the C that is not substituted or is replaced by halogen 1-C 3Alkyl; R 14Hydrogen or methyl; R 15With R 16Be independently selected from hydrogen, methyl and halogen; X is halogen; X ' is halogen or hydrogen; And
In addition, individual R 1, R 2, R 2a, R 3With R 3aBe chosen as hydrogen; Be positioned at two R on contiguous carbon atom 1, R 2, R 2a, R 3With R 3aOptional two keys in conjunction with forming between described contiguous carbon atom; And
Prerequisite is R 1, R 2, R 2a, R 3, R 3a, R4 and R 5In at least one is little electron withdraw group, or R 5Comprise halogen, unsaturated site, carbocyclic ring, heterocycle or azide, unless (ⅰ) R 2With R 2aBe hydrogen, (ⅱ) R 3With R 3aBe hydrogen, (ⅲ) R 1For hydrogen, (ⅳ) between the carbon atom of above-mentioned vicinity, there are two keys, or (ⅴ) R 5For benzyloxymethyl; And
The fucosylation of protein, antibody or the antibody derivatives that wherein generates in body is lower than the protein, antibody or the antibody derivatives that in the situation that lacks the fucose analog, generate in vivo.
In some embodiments of chemical formula (V) and (VI), R 2a, R 3aBe respectively hydrogen.
In some embodiments of chemical formula (V) and (VI), R 5Be selected from lower group :-CH 3,-CH 2CH 3,-CH 2C ≡ CH ,-CH=CHCH 3,-cyclopropyl ,-oxirane, methyl substituted-oxirane ,-CH 2F ,-CH 2Cl ,-CH 2Br ,-CH 2I ,-CHF 2,-CH=C=CH 2,-CH 2N 3With-CH 2CH 2N 3
In some embodiments of chemical formula (V) and (VI), described little electron withdraw group is selected from lower group: fluorine, chlorine, bromine ,-CHF 2,-CH=C=CH 2,-C ≡ CH ,-C ≡ CCH 3,-CH 2C ≡ CH ,-CO 2H ,-C (O) OC 1-C 4Alkyl ,-CH (OAc) CH 3,-CN ,-CH 2CN ,-CH 2X (wherein X is Br, Cl or I), and methyl oxirane.
In some embodiments of chemical formula (V) and (VI), R 5Be selected from lower group :-CH 3,-C ≡ CH ,-CH 2F ,-CH 2Br and-CHF 2.In some further embodiments, R 1, R 2, R 2a, R 3, R 3a, R 4Be selected from independently of one another lower group :-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl.
In some embodiments of chemical formula (V) and (VI), described little electron withdraw group is selected from lower group: fluorine, chlorine, bromine ,-CHF 2,-CH=C=CH 2,-C ≡ CH ,-C ≡ CCH 3,-CH 2C ≡ CH ,-CO 2H ,-C (O) OC 1-C 4Alkyl ,-CH (OAc) CH 3,-CN ,-CH 2CN ,-CH 2X (wherein X is Br, Cl or I) and methyl oxirane.
In some embodiments of chemical formula (V) or (VI), R 1, R 2, R 2a, R 3, R 3aWith R 4In at least two be selected from independently of one another little electron withdraw group.
In some embodiments of chemical formula (V) or (VI), described fucose analog is selected from table 1,2 or 3 compound.
Pharmaceutical composition
The fucose analog of molecule formula I, II, III, IV, V and VI (hereinafter claiming ' fucose analog ') can be prepared the application of being used for the treatment of property.Described fucose analog can be formulated as and comprise treatment or the fucose analog of prevention effective dose and the pharmaceutical composition of one or more pharmacy compatible (can accept) composition.For example, the compositions of medicine or non-medicine generally includes one or more carriers (for example, sterile liquid, for example water and oils, comprise those oil in oil, animal, plant or synthetic source, for example Oleum Arachidis hypogaeae semen, soybean oil, mineral oil, Semen Sesami wet goods).When described pharmaceutical composition was intravenous administration, water was more typical carrier.Saline solution and D/W and glycerite also can be used as liquid-carrier, especially for injection solution.Suitable excipient comprises, for example, aminoacid, starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, Chalk, silica gel, sodium stearate, glyceryl monostearate, Pulvis Talci, sodium chloride, defatted milk powder, glycerol, propylene glycol, ethylene glycol, water, ethanol etc.While needing, described compositions can also comprise a small amount of wetting agent or emulsifying agent, or the pH buffer reagent.These compositionss can have the forms such as solution, suspending agent, Emulsion, tablet, pill, capsule, powder slow release formulation.Description in " Remington ' sPharmaceutical Sciences " (" Lei Mingdun pharmaceutical science ") that the example of suitable pharmaceutical carriers is shown in that E.W.Martin writes.These compositionss contain the mode that the suitable patient of delivering medicine to is provided at the carrier of the described fucose analog for the treatment of effective dose and proper content usually, and contained fucose analog is generally purified form.Described preparation is for administering mode is arranged.
The pharmaceutical composition of describing herein can be any form that allows this compositions is given animal (for example, mammal).The pharmaceutical compositions of describing herein can be to allow to give mammiferous any form by this compositions.The pharmaceutical composition of describing herein can be any form that allows this compositions is given the people.
Described compositions can be the form of solid or liquid.Typical route of administration includes but not limited to, oral, parenteral, Sublingual and eye.Parenteral comprises subcutaneous injection, intravenous injection, intramuscular injection, intrathoracic injection or infusion techn.The preferred parenteral of described compositions or oral giving.These pharmaceutical compositions can be mixed with and make fucose analog when giving animal by described compositions is bioavailable.Compositions also can adopt the form of one or more dosage units, and for example, tablet can be single dosage unit, and the container of solid form fucose analog can hold a plurality of dosage units.
Material for the preparation of described pharmaceutical composition can be nontoxic on its use amount.The optimal dose that those skilled in the art obviously understand the active ingredient in pharmaceutical composition depends on many factors.Correlative factor includes but not limited to, the kind of animal (for example, the mankind), the particular form of fucose analog, administering mode, composition therefor, and the order of severity of pending disease or situation.
Pharmaceutically acceptable carrier or supporting agent can be granular, so that described compositions can be the form of tablet or powder for example.Carrier can be liquid, thereby described compositions is for example oral syrup or injection.
During oral administration, described compositions is preferably solid or liquid form when plan, and is semi-solid herein, semiliquid, and suspending agent, and gel form is included in the solid or liquid range that this paper thinks
As the solid composite for oral administration, described compositions is mixed with powder, granule, compressed tablets, pill, capsule, Chewing gum, the forms such as flat colloid (wafer).The compositions of these solids comprises the diluent of one or more inertia usually.In addition, may have one or more following materials: binding agent is carboxymethyl cellulose, ethyl cellulose, microcrystalline Cellulose or gelatin for example; Excipient is starch, lactose or dextrin for example, disintegrating agent such as alginic acid, sodium alginate, sodium starch glycollate (Primogel), corn starch etc.; Lubricant is magnesium stearate or hydrogenated vegetable oil (Sterotex) for example; Fluidizer is silica colloidal for example; Sweeting agent is sucrose or glucide for example; Flavoring agent is Herba Menthae for example, methyl salicylate or Fructus Citri tangerinae flavor flavoring agent, and coloring agent.
When described compositions was capsule form such as gelatine capsule, outside the above-mentioned type material, it can also contain liquid-carrier, for example Polyethylene Glycol, cyclodextrin or fatty oils.
Described compositions can be liquid form, for example, and elixir, syrup, solution, Emulsion or suspending agent.Described liquid can be sent for oral administration or by injection.When for oral administration the time, compositions can comprise one or more sweeting agents, antiseptic, dyestuff/pigment and flavour enhancer.In the compositions for drug administration by injection (as mentioned above), also can comprise one or more surfactants, antiseptic, wetting agent, dispersant, suspending agent, buffer agent, stabilizing agent and isotonic agent.
Fluid composition, no matter be solution, suspension or other similar type, can also comprise following one or more: the Injectable sterile diluent is water for example, saline solution, preferred normal saline, Ren Shi (Ringer) solution, isotonic sodium chloride, can be used as the fixed oil of solvent or suspension media as synthetic list or double glyceride, Polyethylene Glycol, glycerol, cyclodextrin, propylene glycol or other solvents; Antibacterial such as benzyl alcohol, or methyl parahydroxybenzoate; Antioxidant is ascorbic acid for example, sodium sulfite; Chelating agen such as ethylenediaminetetraacetic acid; Buffer agent is acetate, citrate or phosphate for example, and tension regulator such as sodium chloride or D-glucose.Normal saline is preferred adjuvant.The compositions of injection is preferably aseptic.
As mentioned above, the consumption that can effectively treat the fucose analog of particular disorder or situation will depend on the character of described disorder or situation, and this can determine by the clinical technology of standard.In addition, can adopt alternatively external or in vivo test to assist to identify optimum dosage range.The exact dose that uses in described compositions also depends on route of administration and described disease or the disorderly order of severity, and should decide according to doctor's judgement and each patient's situation.
The fucose analog that described compositions contains effective dose makes and can obtain suitable dosage.Usually, this consumption be the fucose analog account for described composition weight at least about 0.01%.When for oral administration the time, this consumption can described composition weight approximately 0.1% to about 80% scope.Preferred Orally administered composition can contain and accounts for described composition weight approximately 4% to about 50% fucose analog.Preferred composition of the present invention is prepared as and makes the parenteral dosage unit contain to account for the approximately fucose analog of 0.01% to 2% weight.
For intravenously administrable, described compositions can contain approximately 1 to about 250mg fucose analog of per kilogram the weight of animals.In some embodiments, the scope of fucose analog dosage is in about 1-25 mg/kg body weight.The scope of preferred fucose analog dosage is approximately 4 to about 25 mg/kgs of body weight.
Generally, the dosage that gives the fucose analog of animal is that about 0.1mg/kg is to about 250mg/kg with respect to the body weight of described animal usually.Preferably, the dosage that gives animal with respect to the body weight of described animal be about 0.1mg/kg to about 25mg/kg, more preferably the body weight with respect to described animal is that about 1mg/kg is to about 10mg/kg.
The fucose analog that described compositions contains effective dose makes and can obtain suitable dosage.Usually, this consumption be the fucose analog account for described composition weight at least about 0.01%.When for oral administration the time, this consumption can described composition weight approximately 0.1% to about 80% scope.Preferred Orally administered composition can contain and accounts for described composition weight approximately 4% to about 50% fucose analog.Preferred composition of the present invention is prepared as the fucose analog that makes the parenteral dosage unit contain to have an appointment 0.01% to 2% weight.
For intravenous administration, described compositions can comprise approximately 1 to 250mg fucose analog of per kilogram the weight of animals.In some embodiments, the scope of fucose analog dosage is approximately 1 to about 25 mg/kgs of body weight.The scope of preferred fucose analog dosage is approximately 4 to about 25 mg/kgs of body weight.
Generally, according to required effect, the dosage regimen of fucose analog or its pharmaceutical composition can be once a day, and is weekly, biweekly or January once.The administration of fucose analog or its pharmaceutical composition can be from approximately 1 to 5, and approximately 1 to approximately 10, approximately 1 to approximately 15, or more courses for the treatment of, wherein continue one month each course for the treatment of.Giving of each medication course for the treatment of can be once a day, the next day once, biweekly, weekly, biweekly, three weeks once or January once.Can comprise alternatively the rest period course for the treatment of, during the fucosylation of protein (for example antibody or other protein) rise.Alternatively, the rest period can be included between the course for the treatment of.This rest period can make to participate in the fucosylation of the protein of critical function and be replied.
The preferred modes of fucose analog or its pharmaceutical composition is by doctor's tailoring, and will depend in part on the site (for example site of cancer or autoimmune disease) of described medical condition.In one embodiment, described fucose analog or compositions are parenteral.In another embodiment, described fucose analog or compositions are oral administration.
In the specific embodiment, may require one or more fucose analog or compositions part are administered to the zone that needs treatment.This can realize, for example, but is not limited to, and in operation, passes through regional perfusion; Local application; By injection; Perhaps by the implantation means, described implant is porous, non-porous or colloidal material, comprises film, for example silicone rubber membrane, or fiber.In one embodiment, can in cancer, tumor, tumprigenicity or preneoplastic tissue site (or the site in its past), carry out administration by direct injection.
In another embodiment, can in the morbidity site (or the site in its past) of autoimmune disease, carry out administration by direct injection.
In another embodiment, the fucose analog can, at carrier, particularly be sent and (see Langer, Science249:1527-1533 (1990) in liposome; The people such as Treat, at LIPOSOMES IN
THE THERAPY OF INFECTIOUS DISEASE AND CANCER, (infectious disease and
Liposome in treatment of cancer), Lopez-Berestein and Fidler write, when Liss publishes, and New York,
353-365 page (1989); Lopez-Berestein, the same, the 317-327 page; Generally referring to the same).
In another embodiment, fucose analog or compositions can be passed through the controlled release system administration.
In one embodiment, can use pump (referring to Langer, the same; Sefton, CRC Cirt.Ref.
Biomed.Eng.14:201 (1987); The people such as Buchwald, Surgery88:507 (1980); The people such as Saudek, N.Engl.J.Med.321:574 (1989)).In another embodiment, can use polymeric material (referring to MEDICAL APPLICATIONS OF CONTROLLED RELEASE, (" medical application of controlled release ") Langer and Wise write, CRC publishing house, Florida's Boca Raton (1974);
CONTROLLED?DRUG?BIOAVAILABILITY，DRUG?PRODUCT?DESIGN
AND PERFORMANCE, (" controlled medicine bioavailability, the design of medicine and performance ") Smolen and Ball write, Wiley publishing house, New York (1984); Ranger and Peppas, J.
Macromol.Sci.Rev.Macromol.Chem.23:61 (1983); Also can be referring to people such as Levy,
Science228:190 (1985); The people such as During, Ann.Neurol.25:351 (1989); The people such as Howard, J.Neurosurg.71:105 (1989)).Also can use other controlled release systems (Science249:1527-1533 (1990)) of discussing in the summary of Langer.
Term " carrier " refers to diluent, adjuvant or excipient, and the fucose analog together gives with it.This thanks to pharmaceutical carrier can be liquid, and for example water and oils, comprise oil, animal, plant or synthetic those of originating, for example Oleum Arachidis hypogaeae semen, soybean oil, mineral oil, Semen Sesami wet goods.Carrier can be saline, Radix Acaciae senegalis, gelatin, gelatinized corn starch, Pulvis Talci, keratin, silica gel, carbamide etc.In addition, can use adjuvant, stabilizing agent, thickening agent, lubricant and coloring agent.In one embodiment, when giving animal, fucose analog or compositions and pharmaceutically acceptable carrier are aseptic.Water is preferred vector in intravenously administrable.Saline solution and D-glucose aqueous solution and glycerol solution also can be used as liquid-carrier, in particular for injection.Suitable pharmaceutical carriers also comprises excipient, such as starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, Chalk, silica gel, sodium stearate, glyceryl monostearate, Pulvis Talci, sodium chloride, defatted milk powder, glycerol, propylene, ethylene glycol, water, ethanol etc.While needing, compositions of the present invention also can contain a small amount of wetting agent or emulsifying agent, or the pH buffer agent.
With the fucose analog, reduce in vivo cancer, autoimmune disease or the infectious disease that the fucose analog (hereinafter claim ' fucose analog ') of Therapeutic Method molecule formula I provided in this article, II, III, IV, V and the VI of antibody and other albumen fucosylations can be used for treating animal.
The treatment of cancer
The fucose analog can be used for treating patient's cancer.To there being the animal (for example, mammal such as the mankind) that needs to give the fucose analog, can cause the inhibition to the propagation of tumor cell or cancerous cell, or the treatment of the middle cancer of described animal (for example, human patients).The fucose analog can be used for the treatment of the animal cancer by multiple setting.
The fucose analog also can be used for improving interior generation of body of the antibody that lacks the core fucosylation.Raising for the shared ratio of this antibody-like of cancer target in the patient can cause the inhibition to the propagation of tumor cell or cancerous cell, or the treatment of the middle cancer of animal (for example, human patients).The fucose analog can be used for the treatment of the animal cancer by multiple setting.
The particular type of the cancer that can treat with the fucose analog comprises entity tumor and malignant hematologic disease.these cancers include but not limited to: (1) entity tumor, include but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordome, angiosarcoma, hemangioendothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, the bone Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, renal carcinoma, pancreatic cancer, osteocarcinoma, breast carcinoma, ovarian cancer, carcinoma of prostate, esophageal carcinoma, gastric cancer, oral cancer, rhinocarcinoma, laryngeal carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, bone marrow cancer, bronchogenic carcinoma, renal cell carcinoma, the hepatocarcinoma cancer of biliary duct, choriocarcinoma, the spermatogonium cancer, embryonal carcinoma, the WilmShi tumor, cervical cancer, carcinoma of urethra, carcinoma of testis, small cell lung cancer, bladder cancer, pulmonary carcinoma, cell carcinoma, glioma, glioblastoma multiforme, the pleomorphism astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, the auditory nerve tumor, oligodendroglioma, meningioma, skin carcinoma, melanoma, neuroblastoma, and retinoblastoma, (2) haematogenous cancer, include but not limited to: acute lymphoblastic leukemia " ALL ", the acute lymphoblastic B cell leukemia, acute lymphoblastic T chronic myeloid leukemia, acute cellulous leukemia of bone marrow " AML ", acute promyelocytic leukemia " APL ", acute monocytic leukemia, Di Guglielmo syndrome, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocytic leukemia, acute undifferentiated type leukemia, chronic cellulous leukemia of bone marrow " CML ", chronic lymphocytic leukemia " CLL ", hairy cell leukemia, multiple myeloma, acute and chronic leukemia, for example, lymphoblast myelogenous leukemia and lymphatic medullary cell leukemia, and (3) lymphoma lymphogranulomatosis for example, the non-Hodgkin′s lymphomas, multiple myeloma, Fahrenheit (Waldenstrom) macroglobulinemia, heavy chain disease and polycythemia vera.
The multi-modal treatment of cancer
Cancer, include but not limited to: tumor, metastatic carcinoma, perhaps any disease or the disorder take uncontrolled cell proliferation as feature, can pass through above-mentioned Formula I, II, III, IV, any fucose analog of V or VI have the animal (for example, mammal such as people) that needs treat or prevent.In some embodiments, the method for the invention provides is treated or prophylaxis of cancer, and described method comprises the animal that the fucose analog of effective dose and optional chemotherapeutics is had to needs.In one embodiment, described chemotherapeutics is to be used for the treatment of not find it is the cancer of refractory.In another embodiment, described chemotherapeutics is to be used for the treatment of the cancer that is considered to refractory.Can give the fucose analog in order to treat described cancer, to live through the animal of operation.
In one embodiment, additional Therapeutic Method is radiation treatment.
In a specific embodiment, fucose analog and chemotherapeutics or give simultaneously with radiation treatment.In another specific embodiment, the giving of chemotherapeutics or radiation treatment implemented before or after the giving of fucose analog, preferably before or after the administration of fucose analog at least 1 hour, 5 hours, 12 hours, one day, one week, two weeks, three weeks, one month or some month (for example, reaching three months most) carry out.
The administration of chemotherapeutics can continue a series of phase sections (session), and can be the combination of any or chemotherapeutics provided herein.About radiation, can use any radiation treatment scheme according to cancer types to be treated.For example, but be not limited to, can give the X ray radiation; Particularly, high energy megavolt (greater than the radiation of 1 megavolt energy) can be used for deep tumor, and electron beam and the radiation of middle voltage X ray can be used for skin carcinoma.Also can give gamma ray emission type radiosiotope, for example the emission type isotope of radium, cobalt or other elements.
In addition, the invention provides and with the fucose analog, as the substitute of chemotherapy or radiotherapy, treat the method for cancer, be applied to verified or can prove that chemotherapy or radiotherapy toxicity for object to be treated is excessive, for example, the occasion that causes unacceptable or intolerable side effect.Alternatively, the animal of receiving treatment can for example be performed the operation with another kind of cancer therapy, and radiotherapy or chemotherapy are treated, and this depends on that any therapy is considered to acceptable or sustainable.
The multiple medicines thing therapy of cancer
The present invention comprises the method for the treatment of cancer, and the method comprises fucose analog and the therapeutic agent that gives effective dose to the animal that needs are arranged, and this therapeutic agent is anticarcinogen.Suitable anticarcinogen includes but not limited to: methotrexate, and paclitaxel, L-asparaginase, purinethol, thioguanine, hydroxyurea, cytosine arabinoside, cyclophosphamide, ifosfamide, nitroso ureas,
Cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine (procarbizine), hycamtin, chlormethine, cyclophosphamide, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecine, bleomycin, doxorubicin, idarubicin (idarubicin), daunorubicin, D actinomycin D, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel and docetaxel.in a preferred embodiment, described anticarcinogen includes but not limited to: alkylating agent, chlormethine (cyclophosphamide, ifosfamide, trofosfamide, chlorambucil), nitroso ureas (carmustine (BCNU), lomustine (CCNU)), alkylsulfonate (busulfan (busulfan), treosulfan (Treosulfan)), Triazenes (dacarbazine), contain platinum compounds (cisplatin, oxaliplatin, carboplatin), plant alkaloid (vinca alkaloids-vincristine, vinblastine, vindesine, vinorelbine), taxanes (paclitaxel, docetaxel), the DNA topoisomerase enzyme inhibitor, epipodophyllotoxin class (etoposide, teniposide, hycamtin, 9-aminocamptothecin, camptothecine), crisnatol (Crisnatol), mitomycin (ametycin), antimetabolite such as anti-folic acid: dihydrofolate reductase (DHFR) inhibitor: methotrexate, trimetrexate (Trimetrexate), IMP dehydrogenase inhibitor: mycophenolic acid, thiazole furan quinoline (Tiazofurin), ribavirin, EICAR, ribonucleotide reductase inhibitors: hydroxyurea deferoxamine, pyrimidine analogue: uracil analogues: 5-fluorouracil, floxuridine, doxifluridine, Raltitrexed (ratitrexed), cytosine analog: cytosine arabinoside (araC), cytarabin, fludarabine, purine analogue: purinethol, thioguanine, hormonotherapy: receptor antagonist: anti-estrogens: tamoxifen, raloxifene, megestrol, gonadotropin releasing hormone (LHRH) agonist: Gus's crin (goscrclin), leuprorelin acetate, anti-androgens: flutamide (flutamide), bicalutamide, retinoid (Retinoid/Deltoid): vitamin D 3 analogs: EB1089, CB1093, KH1060, optical dynamic therapy agent: Verteporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, nor-oxygen hypocrellin A (2BA-2-DMHA), cytokine class: interferon-' alpha ', interferon-γ, tumor necrosis factor: other: isoprenylation inhibitor, lovastatin, dopaminergic nerve toxin: 1-methyl-4-phenylpyridinium ion, cell cycle inhibitor: D-82041 DEISENHOFEN, D actinomycin D: actinomycin D, dactinomycin, bleomycin class: Bleornycin A2, Bleomycin B2, peplomycin, anthracycline: daunorubicin, doxorubicin (amycin), idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone, MDR inhibitor class: verapamil (Verapamil), and Ca 2+atpase inhibitor class: thapsigargin.
The complementary therapy of cancer
The fucose analog can be used as adjuvant, with the cancer vaccine coupling.Term used herein " cancer vaccine " refers to by inducing and/or strengthening the complex that carrys out the selective destruction tumor cell for the specific immune response of tumor cell.For example, cancer vaccine can be the medicine that comprises the protein of TAA or TSA or peptide, polypeptide, and comprises the pharmaceutical composition of the protein of TAA or TSA or peptide, polypeptide.As described herein, TSA refers to a kind of " tumor-specific antigen " and TAA refers to tumor associated antigen.TSA is the exclusive molecule of cancerous cell.TAA is total in cancerous cell and normal cell but the molecule of differential expression.
The dosage of cancer vaccine can be determined according to the immunoreactive degree that activates for vaccine after suitably adjusting.Usually, as active component, it between 0.01 to 100mg/ days/adult, or preferably between 0.1 with 10mg/ days/adult.Cancer vaccine give can from a couple of days once to the several months once.Can carry out administration according to the medication of knowing of medical peptide, polypeptide or protein, for example subcutaneous intravenous or intramuscular.In order to induce and/or immunoreation while strengthening administration, described peptide, polypeptide or protein can have or not have proper adjuvant while using, with or without with being connected of carrier.Carrier is restriction not, as long as itself does not apply illeffects to human body, and can enhancement antigen; Cellulose, polyamino acid, albumin etc. can be used as the example of carrier.Adjuvant can be to be generally used for those of peptide vaccine inoculation, incomplete Freunds adjuvant, (FIA), and aluminium adjuvant (ALUM), pertussis vaccine, the mineral wet goods can be used as example.In addition, described preparation can suitably be selected by applying the suitable well-known process be used to preparing peptide, polypeptide or protein.
In addition, can also pass through from collecting part mononuclear cell patient's peripheral blood, these cells are hatched together with peptide of the present invention, polypeptide or protein, then will be wherein observe CTL inducing and/or the defeated time patient's of this part mononuclear cell fragment of the activation of CTL blood in.The fucose analog can heavily give the described monocytic while or combine and give thereafter.Condition of culture, monocytic concentration for example, the concentration of described peptide, polypeptide or protein, incubation times etc. can be determined by simply repeating.In cultivation process, can add the material that can strengthen lymphocyte growth, for example interleukin-2.
The treatment of autoimmune disease
The fucose analog can be used for regulating and controlling autoimmune disease or is used for the treatment of autoimmune disease, so that mitigation symptoms and/or autoimmune reaction.The fucose analog can be used for the treatment of by multiple setting the autoimmune disease of animal.
In one embodiment, the autoimmune antibody that the fucose analog is regulated downwards or regulation and control downwards are associated with specific autoimmune disease.
The particular type of the autoimmune disease that can treat with the fucose analog includes but not limited to: Th2-lymphocyte relevant disease (for example, atopy (atopic) dermatitis, atopic asthma, the nose conjunctivitis, allergic rhinitis, Europe Men Shi (Omenn) syndrome, systemic sclerosis, and graft versus host disease); Th1 lymphocyte relevant disease (for example, rheumatoid arthritis, multiple sclerosis, psoriasis, dry (Sjorgren) syndrome, struma lymphomatosa, Robert Graves (Grave) disease, primary biliary cirrhosis, Wegner granulomatosis, and pulmonary tuberculosis); Activated b-lymphocyte relevant disease (for example, systemic lupus erythematosus, goodpasture syndrome (Goodpasture's syndrome), rheumatoid arthritis, and type Ⅰ diabetes mellitus); And following disease.
chronic active hepatitis, Addison disease, sequoiosis, anaphylaxis, allergic rhinitis, Alport syndrome, anaphylactic reaction, ankylosing spondylitis, antiphospholipid syndrome, arthritis, ascariasis, aspergillosis, atopic hypersensitivity, atrophic (atropic) dermatitis, the atrophic rhinitis, behcets disease, the bird-breeder's lung disease, bronchial asthma, rheumatoid pneumoconiosis, cardiomyopathy, celiac disease, South American trypanosomiasis, chronic glomerulonephritis, the sweet syndrome of section, cold agglutinin disease, congenital rubella infects, the CREST syndrome, Crohn disease, cryoglobulinemia, hypercortisolism, dermatomyositis, discoid lupus erythematosus, dressler's syndrome, Eaton-Lambert myasthenic syndrome (Eaton-Lambert Syndrome), echovirus infects, encephalomyelitis, endocrine ophthalmocace, the Epstein-Barr viral infection, broken wind, the yabbi skin ulcer, the Yi Wen Cotard, Felty syndrome, fibromyalgia, Fuchs (Fuch) cyclitis, gastratrophia, gastrointestinal tract allergy, giant cell arteritis, glomerulonephritis, goodpasture syndrome, graft versus host disease, Graves disease, the Guillain Barre disease, struma lymphomatosa, hemolytic anemia, anaphylactoid purpura, idiopathic adrenal atrophy, idiopathic pulmonary fibrosis, IgA nephropathy, inflammatory bowel, insulin dependent diabetes mellitus (IDDM), property childhood arthritis, juvenile diabetes (I type), Eaton-Lambert myasthenic syndrome, laminitis, lichen planus, lupoid hepatitis, the systemic lupus erythematosus lymphopenia, Meniere, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyglandular syndrome, degenerative brain disorder, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, arthritic psoriasis, Raynaud's phenomenon, recurrent abortion, Reiter syndrome, rheumatic fever, rheumatoid arthritis, mountain Te Shi (Sampter) syndrome, schistosomicide, Schmidt syndrome, scleroderma, eosinophilic fasciitis, house Ge Linshi (Sjorgen) syndrome, the stiff man syndrome, sympathetic ophthalmia, systemic lupus erythematosus (sle), Takayasu arteritis, temporal arteritis, thyroiditis, thrombocytopenia, thyrotoxicosis, the Toxic epidermal necrolysis Type B, insulin resistant type type Ⅰ diabetes mellitus, ulcerative colitis, the uveitis vitiligo, Waldenstrom's macroglobulinemia, and wegener granulomatosis.
The multiple medicines thing therapy of autoimmune disease
The present invention also provides the Therapeutic Method of autoimmune disease, and the method comprises to the animal that needs are arranged (for example, mammal) and gives the fucose analog of effective dose and optionally become known for treating the therapeutic agent of autoimmune disease.In one embodiment, this anti-autoimmune disease medicament includes but not limited to: ciclosporin, Ciclosporin A, Mycophenolate Mofetil (mycophenylate, mofetil), sirolimus, tacrolimus, Embrel, prednisone, azathioprine, methotrexate, cyclophosphamide, prednisone, aminocaproic acid, chloroquine, hydroxychloroquine, hydrocortisone, dexamethasone, chlorambucil, dehydroepiandrosterone, danazol, bromocriptine, meloxicam or infliximab.
The treatment of infectious disease
The fucose analog can be used for strengthening immunoreation or is used for the treatment of infectious disease, and described immunoreation can cause the propagation inhibition of the cell that can cause infectious disease or the raising of killing.The fucose analog is used by the infectious disease that multiple setting is used for the treatment of animal.
In one embodiment, the fucose analog strengthens immunoreation, causes causing the killing or suppress of cell proliferation of infectious disease, or improves it and kill or suppress.
the particular type of the infectious disease of available fucose analogue treatment includes but not limited to, (1) bacterial disease: diphtheria, pertussis, recessive bacteremia, urinary tract infection, gastroenteritis, cellulitis, epiglottitis, tracheitis, adenoid vegetation, retropharyngeal abscess, pustule, ecthyma, pneumonia, endocarditis, suppurative arthritis, pneumonia, peritonitis, bacterial meningitis, acute purulent meningitis, urethritis, cervicitis, proctitis, pharyngitis, salpingitis, epididymitis, gonorrhea, the prunus mume (sieb.) sieb.et zucc. poison, listeriosis, anthrax, nocardiosis, Salmonella infection, typhoid fever, dysentery, conjunctivitis, sinusitis, brucellosis, tularemia, cholera, the Mus epidemic disease, tetanus, necrotic enteritis, actinomycetes mixing anaerobic bacterium infects, the prunus mume (sieb.) sieb.et zucc. poison, relapsing fever, leptospirosis, Lyme disease, rat bite fever, tuberculosis, lymphadenitis, leprosy, chlamydia, chlamydia pneumonia, trachoma, inclusion conjunctivitis, systemic infection, (2) fungal infection: histoplasmosis, coccidioidomycosis, blastomyces, sporotrichosis, cryptococcosis, systemic candidiasis, aspergillosis, mucormycosis, mycetoma, painted mycosis, (3) rickettsiosis: typhus fever, American spotted fever, the Ehrlichia disease, the east Ticks passes rickettsial pox, rickettsiosis, Q heat, and bartonellosis, (4) parasitic disease: malaria, the Babesia disease, lethargus, South American trypanosomiasis, leishmaniasis, kala azar, toxoplasmosis, meningoencephalitis, keratitis, amebiasis, giardiasis, cryptosporidiosis, isosporiasis, the circle sporidiosis, microsporidiosis, ascarid, whipworm infection, hookworm infection, retrofection, the eye larval migration, trichonematosis, Guinea's dracunculiasis, Filariasis, loaiasis, onchocerciasis, the dog heartworm infects, schistosomicide, schistosome dermatitis, oriental lung fluke disease, oriental liver fluke disease, fascioliasis hepatica, fasciolopsiasis buski, opisthorchiasis, cestode infection, echinococcosis, hydatid disease,alveolar, (5) viral disease: measles, subacute sclerosing panencephalitis, common cold, parotitis, rubella, erythema, erythema infectiosum, chickenpox, respiratory syncytial virus infection, croup (Croup), bronchiolitis, infectious monocytosis, poliomyelitis, herpangina, hand-foot-mouth disease, rich En Huoermu disease, genital herpes, genital wart, aseptic meningitis, myocarditis, pericarditis, gastroenteritis, acquired immune deficiency syndrome (AIDS) (acquired immune deficiency syndrome (AIDS)), Reye's syndrome, kawasaki syndrome, influenza, bronchitis, viral action pneumonia (Viral " Walking " Pneumonia), the febris acuta respiratory tract disease, acute pharyngoconjunctival fever, epidemic keratoconjunctivitis, herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), herpes zoster, cytomegalovirus infection, rabies, progressive multifocal leukoencephalopathy, Kuru disease, fatal familial insomnia, gram Ya Shi disease, the GSSShi disease, torrid zone spastic paralysis, Western equine encephalitis, galifornia encephalitis, St. Louis encephalitis, yellow fever, dengue fever, lymphocytic choriomeningitis, lassa fever, hemorrhagic fever, Hantaan virus, the lung syndrome, Marburg virus infects, Ebola virus viral infection and variola.
The multiple medicines thing treatment of infectious disease
The present invention also provides the method for the treatment of infectious disease, comprises the fucose analog and the optional therapeutic agent that to the animal that needs are arranged (for example, mammal), give effective dose, and this therapeutic agent is the medicament for anti-infectious disease.In one embodiment, the medicament of anti-infectious disease includes but not limited to: (1) antibacterial: beta-lactam antibiotic: benzylpenicillin, penicillin V, cloxacillin, dicloxacillin, methicillin, NAFCILLIN, oxazacillin, ampicillin, amoxicillin, bacampicillin, azlocillin, Carbenicillin, mezlocillin, piperacillin, ticarcillin; Aminoglycoside: amikacin, gentamycin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin; Macrolide: azithromycin, clarithromycin, erythromycin, lincomycin, clindamycin; Fourth Ring class: demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline; Quinolones: cinoxacin, nalidixan, fluoroquinolones: ciprofloxacin, enoxacin, grepafloxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, Sparfloxacin, trovafloxacin; Polypeptide class: bacillin, colistin, polymyxin B; Sulfonamides: sulfanilamide is different
Figure BDA00002816630300361
Azoles, sulfalene
Figure BDA00002816630300362
Azoles, sulfadiazine, sulfamethizole, sulfacetamide; Other antibacterial: trimethoprim, sulfanilamide diformazan
Figure BDA00002816630300363
Azoles, chloromycetin, vancomycin, metronidazole, quinupristin, dalfopristin, rifampicin, spectinomycin, furan is appropriate; Antiviral agent: common antiviral agent: Idoxuridire, arabinose adenosine, trifluridine, acyclovir, famciclovir, dissolve VCV (pencicyclovir), valaciclovir, Cymevan (gancicyclovir), FOSCARNET, ribavirin, amantadine, rimantadine, cidofovir; The antisense oligonucleotide class; Immunoglobulins; Interferons; HIV (human immunodeficiency virus) infection medication: zidovudine, Didanosine, Zha Xi its shore, stavudine, lamivudine, nevirapine, delavirdine, Saquinavir, ritonavir, indinavir and viracept see nelfinaivr.
The other treatment agent
Method of the present invention also can comprise and gives fucose analog and therapeutic agent or the acceptable salt of its pharmacy or solvate.Fucose analog and therapeutic agent can play accumulative action, or more preferably play synergism.In a preferred embodiment, contain fucose compositions give with one or more therapeutic agents give carry out simultaneously, these therapeutic agents can be in containing the same compositions of fucose analog, or in another compositions.In another embodiment, the fucose analog give before or after the administration of described therapeutic agent, carry out.
In the method for the treatment of cancer of the present invention, autoimmune disease or infectious disease, therapeutic agent can also be antiemetic.suitable antiemetic includes but not limited to: metoclopramide (metoclopromide), domperidone, prochlorperazine mesylate, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, the acetylleucine monoethanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, marezine, dimenhydrinate, diphenidol, dolasetron, mechlizine, methallatal, metopimazine, nabilone, spray ground, west difficult to understand, pipamazine, scopolamine, sulpiride, tetrahydrocannabinol, thiethylperazine, thioproperazine, and tropisetron.
In another embodiment, described therapeutic agent can be the hematopoietic cell colony stimulating factor.Suitable hematopoietic cell colony stimulating factor includes but not limited to: filgrastim (filgrastim), Sargramostim (sargramostim), molgramostim and erythropoietin α.
In another embodiment, described therapeutic agent can be opiates or non-opium analgesics.Suitable opioid analgesic includes but not limited to: morphine, heroin, hydromorphone, the hydrogenation codeinone, oxymorphone, 14-hydroxycodeinone., Mai Tuopeng, apomorphine, normorphine, etorphine, buprenorphine, pethidine, loperamide, anileridine, ethoheptazine, Mi Niding (piminidine), betaprodine, diphenoxylate, fentanyl, sufentanil, alfentanil, remifentanil, levorphan, dextromethorphan, benzene azoles star, pentazocine, Cyc, methadone, isomethadone and dextropropoxyphene.Suitable non-opium analgesics includes but not limited to: aspirin, celecoxib, rofecoxib, diclofenac sodium, diflunisal (diflusinal), etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, Meclofenamate Sodium, mefenamic acid, nabumetone, naproxen, piroxicam and sulindac.
The present invention has a detailed description in following embodiment, but these embodiment not meaning that limit the scope of the invention.
Embodiment
Embodiment 1: in the body of non--fucosylation antibody, generate
Method:
30-60 days before starting this research, with the immune female BALB/c mouse of keyhole limpet hemocyanin (KLH).In first research, mice continues by ip, to be given once a day in 7 days the alkynyl fucose (SGD-1887) of 150mg/kg, peracetic acid alkynyl fucose (SGD-1890), 2-fluorine fucose (SGD-2083), or 2-fluorine fucose triacetate (SGD-2084) or be not subject to processing.At second day, mice is also used the KLH booster immunization.The last time the next day of ip administration, mice is carried out to end Mo and take a blood sample and obtain serum.In second research, mice continues in its drinking water, accepted the 2-fluorine fucose (SGD-2083) of 100mM in 7 days or accept raw water, use the KLH booster immunization, the drinking water or the raw water that continue afterwards to contain 100mM2-fluorine fucose continue 7 days, then carry out end Mo and take a blood sample.Obtain subsequently serum.In two researchs, all there is no dead mouse.In two researchs, serum all obtains the specific polyclonal antibody of KLH-by commercially available resisting-KLH compatible column.Effluent by anti--KLH compatible column obtains all the other antibody by commercially available a-protein post.
Dot blot: will be from the antibody of unprocessed and treated animal, and as the antibody cAC10 of the known core fucosylation of having of standard amount (0 to 100% fucose), each point sample 0.5 μ g is to nitrocellulose filter.Protein level dyes and manifests by Ponceaux.
Trace is surveyed with biotin labeled aspergillus oryzae L-fucose specific agglutination element (AOL) (can in conjunction with the antibody of fucosylation) and with Streptavidin albumen horseradish peroxidase (HRP) and ECL imaging.Gel loading (visible) and fucose signal (bioluminescence) are measured by Alpha Innotech camera and are quantitative with machine software.
Gas chromatogram (GC): by the antibody from unprocessed and treated animal of water dialysis respectively being got to 40 μ g, carry out Methanol Decomposition in hydrochloric acid methanol.The same antibody cAC10 check sample of processing with 0 to 100% fucose.The gained methylglycoside is by using commercially available mixture Tri-Sil to carry out trimethyl silicone hydride and derivatization to monosaccharide alcohol.Gained trimethyl silane methylglycoside on Hewlett-Packard (Hewlet Packard) gas chromatograph that detects with flame ion in Agilent (Agilent) J& On W DB-1 post, with thermograde, detect.By will relatively identifying Relative Peak with the saccharide of the parallel derivatization of antibody sample is carried out retention time.Peak uses the GC Software Integration.The peak area ratio of fucose/mannose is for determining the fucosylation state of antibody.
Result:
Research 1: Fig. 1 shows the Dot blot (left side) of the antibody be not combined with KLH but regain from the protein A post.Shown result is the cAC10 standard (Dot blot of below, the respective column of leftmost side dotted line frame and top Dot blot), undressed contrast (the Dot blot of below, and alkynyl fucose (SGD-1887 the respective column of second dotted line frame and top Dot blot from left to right); The Dot blot of below, middle dotted line frame and the respective column of top Dot blot), peracetic acid alkynyl fucose (SGD-1890; The Dot blot of below, the respective column of second dotted line frame and top Dot blot from right to left), and 2-fluorine fucose (SGD-2083; The Dot blot of below, the respective column of rightmost side frame and top Dot blot).Proofread and correct the point sample water gaging flat, also the percentage rate of fucosylation is presented in the coordinate diagram of right side.On average, with undressed the contrast, compare, the level of antibody fucosylation has reduced approximately 1/3rd.
Fig. 2 shows from the fucosylation level of the anti-KLH antibody of separating processed group (figure A and B) and all the other serum IgG molecules (figure C and D).The fucosylation level all is shown as the percentage value (figure B and D) based on the fucosylation percentage rate of cAC10 antibody standard (figure A and C) and the relative unprocessed cell mean of processed group meansigma methods.On average, the fucosylation level of anti--KLH antibody was passed through after three kinds of fucose analog are processed to reduce approximately half.All the other antibody that gather also demonstrate the core fucosylation and reduce approximately 1/4th.In this research, the total antibody horizontal (KLH-specificity and non-specific) after mice contact KLH has rising.Therefore, most antibody is new synthetic during processing.These observe the core fucosylation that surface new antibody that produces after the administration of fucose analog can demonstrate reduction.
Research 2: in this research, detect the effect of fucose analog oral administration.Fig. 3 shows by the oral result that gives fucose analogue treatment mice.What detect antibody fucosylation level is with KLH, not to be combined, but those antibody of regaining from the protein A post.Shown result is the cAC10 standard (Dot blot of top and below, leftmost side frame), undressed contrast (the Dot blot of top and below, the frame on second (top) and right side from left to right), and 2-fluorine fucose (Dot blot of top and below, second (below) and second from right to left (top and below) frame from left to right).After correction point sample water gaging is flat, also fucosylated percentage rate is presented in the coordinate diagram of right side.Core fucosylation level from the antibody of treated animal is almost eliminated: on average, the fucosylation level of treated animal be 7% and unprocessed animal when being 81%.These observations show that the oral administration of fucose analog is the effective means that reduces antibody fucosylation level.
Embodiment 2: the external activity of fucose analog in cell culture
Roughly, described in the U.S. Patent application 2009-0317869 that has announced, assessed the impact of fucose analog antagonist core fucosylation under 50 μ M and 1mM concentration.The summary scheme is as follows: will produce humanization IgG1 anti--the CHO DG44 cell line h1F6 (referring to international patent publications WO06/113909) of CD70 monoclonal antibody, in the CHO culture medium of 2 milliliters with 7.5x10 5Every milliliter, individual cell, in 37 °, 5%CO 2And the 100RPM shaken cultivation is in six porocyte culture plates.Culture medium has been added insulin like growth factor (IGF), penicillin, the fucose analog of streptomycin and 1mM or 50 μ M.After inoculation the 5th day, by culture under 13000RPM centrifugal 5 minutes so that cell precipitation; Subsequently from the supernatant antibody purification.
Antibody purification is by being applied to conditioned medium (conditioned media) through 1X phosphate buffered solution (PBS) pre-equilibration, the a-protein resin of pH7.4.After the 1X PBS with 20 times of resin volumes cleans resin, with the pure IgG elution buffer of the immunity of 5 times of resin volumes (Pierre Si biotech company (Pierce Biotechnology), Illinois Rockford) eluting antibody.In elution fraction, add the 1M Tris buffer of 10% volume pH8.0 to make it neutralization.Result is as shown in following form.
Table 1
Figure BDA00002816630300401
Figure BDA00002816630300411
Figure BDA00002816630300421
" ND " refers to detect due to antibody generation difference or cell growth inhibition under the existence of fucose analog.
Table 2
Figure BDA00002816630300422
When " ND " existed while referring to due to the fucose analog, antibody produced poor or cell growth inhibition and detects.
Test some fucose analog and be included into the ability of antibody.These fucose analog are tested with said method under 50uM and 1mM concentration.The results are shown in shown in following table.
Table 3
Figure BDA00002816630300431
Figure BDA00002816630300441
These test for identifications in mammal, suppress the candidate compound of core fucosylation.
Embodiment 3: the generation of non-fucosylation antibody in body after oral administration
In this research, further check the oral effect that gives fucose analog 2-fluorine fucose (SGD-2083).Continue in female BALC/c mice drinking water, to give in 14 days 1,10 with 100mM2-fluorine fucose.Mice is used the classical adjuvant immunity of TiterMAX, and continue in its drinking water, to give in 7 days 1,10 with 100mM2-fluorine fucose.To the whole end of mice, get blood and obtain serum subsequently.By serum is gone out to endogenous antibody by the a-protein column purification.
The antibody of collecting is assessed the fucosylation level by following Dot blot.Will be from the antibody of treated and unprocessed animal (each 0.5 μ g), and with the cAC10 standard (only studying 1) of 0 to 100% fucose, trace is to nitrocellulose filter.Protein level dyes and presents by Ponceaux.Trace is surveyed with biotin labeled AOL agglutinin and with Streptavidin HRP and ECL imaging (as mentioned above).Gel loading (visible) and fucose signal (bioluminescence) are measured and use machine software quantitative by Alpha Innotech photographing unit.
Result:
The fucosylation level has dose dependent to reduce between three kinds of concentration of 2-fluorine fucose (SGD-2083).Referring to Fig. 4, the fucosylation level of antibody the highest in the 2-of undressed matched group and 1mM fluorine fucose group (left figure and middle figure, two of tops frame).The almost Ex-all of fucosylation of the antibody of 2-fluorine fucose moderate concentration group, close with the 100mM2-fluorine fucose group of high concentration.These results confirm that to mice, giving 2-fluorine fucose can suppress the core antibody fucosylation.
Embodiment 4: the impact of fucose analog on people's cell
Study different fucose analog and suppressed the ability that human myeloma cells produce the fucosylation of the ability of fucosylation of IgG antibody and inhibition human carcinoma cell line surface protein.In first research, the ability of investigating the fucosylation of fucose analog inhibition cell line IgG that LP-1 produces obtains, and this cell line is people's multiple myeloma cell line.The antibody that undressed LP-1 cell produces and finds in its culture medium turns out to be IgG type (data do not show) by the Western blotting that detects with Anti-Human IgG.This is by (250,000 thin by the LP-1 cell of 20mL in the T-75 culture bottle
Born of the same parents/milliliter) in 37 ° of C, containing in the humid atmosphere of 5%CO2, cultivation realized over 5 days in the tissue culture medium (TCM) (the hot deactivation FBS that 90%RPMI and 10%IgG exhaust) that IgG exhausts.By centrifugal cell harvesting (200x g, 4 ° of C, 5 minutes), and collect culture medium.Culture medium is filtered by 0.22 μ m filter, with 1mL, contains 50%MabSelect subsequently TMThe PBS of a-protein paste resin catches IgG 4 ° of C rotation overnight incubation.Make the paste resin sedimentation and remove most of culture medium.By in media transfer to two a cellulose acetate filtering centrifuge tube of paste resin with about 0.5mL, and with 5000x g centrifugal 1 minute.Subsequently resin bed is cleaned 3 times with the PBS of 0.5mL.With the PierceIgG elution buffer eluting IgG (adding the 9M Tris buffer of 52 μ L pH9.5 after eluting to regulate pH) of 700 μ L, wash out IgG.The gained eluent is transferred to cutoff value 10, the centrifugal concentrating device of 000MW, by sample concentration to about 20 μ L.The concentrated sample of 1 μ L is loaded on sds polyacrylamide glue and separates then trace to the nitrogen cellulose membrane.Ponceaux is used in trace dyeing for total protein, and identifies isotype with Anti-Human IgG antibody.It is consistent with heavy chain and the light chain molecular weight of IgG that total protein dyeing demonstrates band, and the dye reaction that demonstrates protein band of Anti-Human IgG is consistent with the molecular weight of heavy chain, as expection.
The antibody fucosylation also can use biotin labeled aspergillus oryzae L-fucose specific agglutination element (AOL) to determine, the α-1,6 of AOL specific binding antibody links fucose.This fucose detection method for by SDS-PAGE, separated or without separation, be loaded to trace albumen on nitrocellulose filter and all be suitable for.Use AOL-biotin conjugates and Streptavidin-HRP and ECL to detect the fluorescence signal that produces and can use Alpha Innotech
Figure BDA00002816630300461
Q system is come quantitatively.As expection, separate from the IgG of LP-1 culture and in the band of corresponding heavy chain molecule amount (MW), show that AOL-relies on signal (data do not show).Analog 2-fluorine fucose (SGD-2803) and 2-fluorine fucose peracetic acid ester (SGD-2804) do not demonstrate the fucosylation of antibody, but alkynyl fucose peracetic acid ester (SGD-1890) has shown.
In order further to assess the activity of different fucose analog, tested 48 kinds of different fucose analog and the other four kinds of glycosylation inhibitors capability of influence to the fucosylation of IgG that LP-1 produces.LP-1 cell (250,000 cell/mL, each compound 3mL in 6 well culture plates) with each fucose analog one of 100 μ M, arise from 37 ° of C in containing the humid atmosphere of 5%CO2, in the tissue culture medium (TCM) (the hot deactivation FBS that 90%RPMI and 10%IgG exhaust) that IgG exhausts, hatched 5 days.As mentioned above, each sample only uses the MabSelect of 0.5mL in the separation of IgG TMA-protein paste resin, and a centrifuge tube, the eluent of 400 μ LIgG elution buffers add the 9M Tris buffer of 25 μ LpH9.Eluent is concentrated into each sample 10-20 μ L, through concentrated eluent, respectively gets 2 μ L point samples and dye to judge and regulate the sample loading for AOL dyeing to nitrocellulose filter and with Ponceaux.According to this judgement to total protein in each sample, each sample is got approximately 0.5 μ g point sample and, to film, is dried, and dye with Ponceaux.The image of this dyeing film uses Alpha Innotech
Figure BDA00002816630300462
Q system obtains.With 5% bovine serum albumin (BSA) sealing 1 hour, with after TBST (TBS contains triton) washing 3 times, and hatched together 1 hour by 5 μ g/ml biotinylation AOL in Tris buffer saline (TBS) for this film subsequently.Film is washed 3 times with TBST again, then with Streptavidin-HRP, hatched 30 minutes and wash 3 times with TBST is last.The bioluminescence signal uses chemical illuminating reagent (ECL) to manifest and uses Alpha Innotech
Figure BDA00002816630300463
Q system with
Figure BDA00002816630300464
Software analysis.The results are shown in shown in following table.For some analog, a plurality of samples have been analyzed as shown in Table.
Table 4
The molecule title The SGD numbering The fucosylation of IgG and the fraction values that contrasts
The alkynyl fucose 1887 3
Alkynyl fucose peracetic acid ester 1890 0；0.08；2
5-vinyl fucose tetracetate 1922 2
5-cyano group methylene fucose tetracetate 1924 96
L-GaA-Isosorbide-5-Nitrae-lactone 1931 81
Methyl a-L-rock algae pyranoside 1932 87
5-propinyl fucose tetracetate 1937 315
5-(Z)-acrylic fucose peracetic acid ester 1944 72
The amino fucose of 6-propargyl 1950 40
5-methyl ester fucose tetracetate 1959 94
Castanospermine 1960 300
5-methyl ketone fucose tetracetate 1964 5
6-bromine fucose tetracetate 1969 1
5-isopropyl fucose tetracetate 1977 79
Several husband's alkali (kifunensine) 1978 0.44；2
Propargyl fucose tetracetate 1987 38
6-fluorine fucose tetracetate 1988 15
5-ethyl fucose tetracetate 1989 11
5-carboxylic amine fucose tetracetate 1995 79
6-alkynyl-6-acetoxyl group fucose tetracetate 2004 29
Alkynyl fucose four propionic esters 2010 1.5
Alkynyl fucose four alkyl caproates 2012 67
5-epoxy fucose tetracetate 2020 5
6-sulfur lactose pentaacetate 2025 44
Alkynyl fucose four isobutyrates 2043 34
6-formyl fucose tetracetate 2045 70
6'6-difluoro fucose tetracetate 2046 2
Alkynyl fucose four nicotinates 2047 50
Benzyloxy fucose tetracetate 2048 114
Alkynyl fucose four PEG esters 2057 64
Alkynyl fucose four iso-nicotinates 2058 34
1-methyl alkynyl fucose triacetate 2059 89
6-carboxymethyl ester fucose tetracetate 2061 71
6-ketone-6-ethyl fucose tetracetate 2067 3
5-(2-cyano ethyl) arabinose tetracetate 2070 51
D-galactose pentaacetate 2074 118
1,2-dideoxy-1,2-dehydrogenation fucose diacetate esters 2080 159
1-abequose triacetate 2081 70
1,2-difluoro fucose diacetate esters 2082 87
The fluoro-2-abequose of 2- 2083 66
The fluoro-2-abequose of 2-tetracetate 2084 52
6-allene fucose tetracetate 2097 45
The chloro-2-abequose of 2-tetracetate 2099 146
2-abequose triacetate 2108 104
3-sulfur fucose tetracetate 2112 64
Angustose 2113 93
4-abequose triacetate 2134 49
Select 3 kinds of fucose analog to carry out comprehensive SDS-PAGE/ western blot analysis and show that this technology can detect the variation of fucose signal on heavy chain.Selected three kinds of analog use under 50 μ M concentration.2-fluorine fucose (SGD-2083) has been compared in these analyses, 2-fluorine fucose peracetic acid
Ester (SGD-2804), and alkynyl fucose peracetic acid ester (SGD-1890) is to the activity of the antibody from unprocessed cell.The IgG that uses alkynyl fucose peracetic acid ester to produce does not show the reactivity to biotin labeled AOL, confirm that the method can detect the variation of AOL signal, and 2-fluorine fucose (SGD-2083) has no significant change with the AOL signal of 2-fluorine fucose peracetic acid ester (SGD-2804).These results conform to the result of these compounds in table 4 substantially.
In table 4, a lot of fucose analog of test be it seems the fucosylation that reduces the antibody that human myeloma cell produces.As the indication that suppresses, the Dot blot of described compound shows that wherein 10 kinds is the potential potent inhibitor of IgG fucosylation in the human cell with the AOL signal weakening.These fucose analog are: alkynyl fucose peracetic acid ester (SGD-1890), alkynyl fucose four propionic esters (SGD-2010), 1 – methyl fucose triacetate (SGD-2039), 5--ethyl fucose tetracetate (SGD-1989), 6-fluorine fucose tetracetate (SGD-1988), 6-bromination fucose tetracetate (SGD-1969), 6'6-difluoro fucose tetracetate (SGD-2046), 6-ketone-6-ethyl fucose tetracetate (SGD-2067), 5-epoxy fucose tetracetate (SGD-2020), and 5-methyl ketone fucose tetracetate (SGD1964).
For the result of further clear and definite AOL Dot blot, to following fucose analog: alkynyl fucose peracetic acid ester; 5-vinyl fucose tetracetate; 5-methyl ketone fucose tetracetate; 6-bromine fucose tetraacethyl; 6-fluorine fucose tetracetate; 5-ethyl fucose tetracetate; 5-epoxy fucose tetracetate; 6'6-difluoro fucose tetracetate; 6-ketone-6-ethyl fucose tetracetate (SGD-2067) and 2-fluorine fucose peracetic acid ester, isolate the sample of the IgG that processed cell produces (the AOL signal of Dot blot demonstrates medium to strongly weakening) and with reduced form PLRP-MS, check molecular weight (MW) the confirmation fucosylation state that utilizes heavy chain.
As mentioned above, by the LP-1 cell sample of 40mL (250,000 cells/mL) processed 5 days with the fucose analog of 100 μ M, and use as previously mentioned a-protein resin purification IgG.Yield uses the UV optical spectroscopy to detect, and supposes that extinction coefficient are 1.4AU/ (mg/mL).Seven in ten compounds have obtained enough IgG and have analyzed (use SGD-2067, the IgG that SGD-1964 and SGD-2020 produce<10 μ g, may be due to the toxicity of these analog to cell).All the other IgG 37 ° of C reduction 15 minutes and with the PLRP separation, use the QTOF mass spectrograph to carry out the MS analysis with 10mM DTT subsequently.Detect the heavy chain peak that obtains, and compare with the IgG that undressed cell produces.
The results are shown in Table shown in 5 (following tables) of mass spectrography.Heavy chain deducts fucose with respect to heavy chain and heavy chain deducts the peak heights that fucose adds fucose analog (may improve if analog is included into the antibody saccharide) by comparing, and assesses mass signal.In ten test-compounds, there are four to be on antibody 1, the partially or completely inhibitor of 6-fucosylation.Alkynyl fucose peracetic acid ester (SGD-1890) provides fully and suppresses, and 2-fluorine fucose (SGD-2084) to suppress 70% be suboptimum, be secondly 6'6-difluoro fucose tetracetate (SGD-2046) and 6-bromine fucose tetracetate (SGD-1969) inhibition approximately 33 and 20% and include saccharide in described analog.
Table 5.
The PLRP-MS result of IgG that LP-1 produces and Dot blot result
Figure BDA00002816630300491
Embodiment 5: the impact of fucose analog on the protein fucosylation
For human cancer cell, test four kinds of partially or completely inhibitor: alkynyl fucose peracetic acid ester (SGD-1890), 2-fluorine fucose peracetic acid ester (SGD-2084), 6'6-difluoro fucose tetracetate (SGD-
2046), with 6-bromine fucose tetracetate (SGD-1969) on the impact of cell surface fucosylation, by five kinds of different people source cancerous cell lines (Caki-1, PC-3, Ramos, LS174t, and HL60cy) hatch test.The various inhibitor of 100 μ M use about 1-2 week under the standard culture environment, during periodic replacement comprise the culture medium of fresh inhibitor.After incubation period, use four kinds of different detectable: biotinylation lens culinaris agglutinin-A (LCA), anti--Lewisx antibody (anti--SSEA1), anti--Lewisy antibody (cBR96) and recombined human palatelet-selectin/CD62P/Fc fusion rotein.Then process comprises hatches cell 1 hour in 4 ° of C with the one-level detectable for 3 times with FACS buffer (PBS+10% bSA+0.02% sodium azide) washing, then with after FACS buffer solution for cleaning 3 times, with 4 ° of C of secondary detection reagent, hatches 1 hour.Finally cell also again is suspended in the FACS buffer, and detects with BD FACScan instrument for 3 times with the FACS buffer solution for cleaning.The α of the N-acetylchitobiose part that LCA reagent identification is incorporated into core oligosaccharide with sequence and its affinity of α-connecting-type mannose residue-connecting-type fucosyl residues significantly improves.The palatelet-selectin fusion rotein detects the palatelet-selectin part that is positioned at cell surface, and this interaction relates to the Slex epi-position on the palatelet-selectin part.
Detected cell line all shows the LCA reagent dyeing, and this reagent identification contains the sequence of α-connecting-type mannose residue, and its affinity is incorporated into the α of the N-acetylchitobiose part of core oligosaccharide-connecting-type fucosyl residues and significantly improves.The LCA of this sugared epi-position detects after using all inhibitor (100 μ M) to process cell and reduces.This prompting fucose is used the impact of six kinds of fucose analog processing that detect in the overall existence of cell surface.
Fig. 7 shows the result of above-mentioned research.For Lewis X, in the cell line that detects, only have undressed LS1745t and HL60cy Lewis obviously to be detected at cell surface X(anti--SSEAI dyeing) (Fig. 7 A).Resisting-SSEAI detection all significantly reductions after using fucose analog (100 μ M) to process cell of this structure.
For Lewis Y, in the cell line that detects, only have undressed LS1745t and HL60cy Lewis obviously to be detected at cell surface Y(cBR96 dyeing) (Fig. 7 A).The cBR96 of this structure detects after using all fucose analog (100 μ M) to process cell and significantly reduces.
For palatelet-selectin, in the cell line that detects, only have undressed HL60cy the palatelet-selectin part obviously to be detected at cell surface.The detection of this part all decreases after using all fucose analog (100 μ M) to process cell, except alkynyl fucose peracetic acid ester (SGD-1890) processing (Fig. 7 C).Undressed Ramos cell demonstrates few palatelet-selectin part; Yet the signal of this part strengthens after using the fucose analog to process.This is uncommon and does not observe in the past when to these cells, using 2-fluorine fucose (SGD-2083) or alkynyl fucose (SGD-1887) to process.
Result shows with these fucose analog to be processed and can affect generally the fucose of cell surface and also affect specifically cell surface Lewis XAnd Lewis YFucosylation and the sialyl Lewis X on the palatelet-selectin part modified.
Embodiment 6: reduction is combined in the leukocytosis after 2-fluorine fucose oral administration with E-Selectin
In mice, detect the impact that the fucose analog is combined with E-Selectin on leukocytosis.Female Balb/c mice is accepted (SGD-2083) administration of oral 2-fluorine fucose or does not process in drinking water.Mice was taken a blood sample before administration, three weeks took a blood sample weekly once afterwards, carried out assessments cell quantity and they ability in conjunction with E-Selectin.In a research, 2-fluorine fucose is formulated in (every group of n=3) in drinking water with 1mM, 10mM or 100mM.At the 14th day, use
Figure BDA00002816630300511
Classical adjuvants (Sigma company (Sigma)) are processed mice to activate polyclonal, the antigen non-specific antibody that is produced by the B cell, and continue to contain the drinking water to the 21 days of 2-fluorine fucose.In second research, mice continue to accept in quoting water in 3 weeks to be mixed with 10mM and 100mM 2-fluorine fucose and without any other processing (n=6).At the 21st day, beyond blood, also detect the lymph node pool (armpit, humerus, shallow table groin and mesentery) that comes from three animals.By lymph node homogenate single cell suspension, and determine total cell number by counting on hematimeter, with trypan blue, get rid of dead cell.In order to determine the total leukocyte quantity of every microliters of blood, will on hematimeter, count from the blood sample of individual animals, use Turk solution (3% acetic acid that contains 0.01% Gentian Violet) to get rid of erythrocyte (RBC).In order to carry out flow cytometry, by infiltration, dissolve from residual blood, removing erythrocyte.Cell hatches to identify neutrophil cell together with anti--Gr-1-FITC antibody (BD Biological Science Co., Ltd), and with restructuring E-Selectin-people Fc fusion rotein (R& D system company) hatch.Cell is through hatching to detect the E-selectin of combination together with the goat Anti-Human IgG-Fc secondary antibody (Jackson's immune Research company (Jackson Immunoresearch)) of PE-labelling after washing.The CellQuest software analysis is collected and used to sample through the FACSCalibur flow cytometer.Determine the percentage rate of Gr-1+ cell, and with hematimeter counting gained total leukocyte number, calculate the absolute quantity of neutrophil cell.In addition, the sample of fluidic cell establishes to the Gr-1+ cell combination that gate estimates by histogram analysis E-Selectin and neutrophil cell.By rectangular histogram, determined the geometrical mean of E-Selectin fluorescence signal.
Result
The result of Fig. 5 A and 5B shows that the oral administration of 2-fluorine fucose (SGD-2083) causes the leukocyte that circulates and neutrophil cell to raise in the dose dependent mode.The 2-fluorine fucose administration effect of 1mM is minimum, and along with 2-fluorine fucose dosage is brought up to 10mM and 100mM, the effect of observing strengthens.Data shown in Fig. 5 A and 5B come from the 14th day of first research.The 7th and 21 days and second research of first research the 714th and also observed similar result (data do not show) in 21 days.In second research, also assess the lymph node of the 21st day and in Fig. 5 C, show 2-fluorine fucose oral administration cause lymph node in cellularity significantly reduce.With 10mM, compare, the effect of 100mM is more violent.
2-fluorine fucose oral administration also causes the combination of E-Selectin and neutrophil cell to reduce (Fig. 6).The effect of fucose analog is also dose dependent, and the effect of 1mM is minimum, and the effect of 10mM and 100mM strengthens (Fig. 5 B and Fig. 5 C).
The circulation leukocyte of observing is suppressed consistent in conjunction with E-Selectin with rising (leukocytosis) and the neutrophil cell of neutrophil cell.The E-Selectin mediated leucocytes is seeped into outward in peripheral blood and lymph node, and the inhibition of E-Selectin combination (by suppressing fucosylation) also can reduce and exosmoses and cause the leukocyte accumulation in blood.The fucose analog of these results suggest energy Profilin matter fucosylations, particularly E-Selectin fucosylation can suppress autoimmune.
Embodiment 7: by giving the fucose analog, suppress tumor growth
Research 1
At external evaluator source cell, be the sensitivity to fucose analog 2-fluorine fucose.Cell line used is: the LS174T adenocarcinoma of colon, and the PC-3 adenocarcinoma of colon, the HL-60 acute myeloblastic leukemia,
Ramos burkitt's lymphoma, and Caki-1 renal cell carcinoma.Cell line is being added 100 μ M's
In the culture medium of 2-fluorine fucose (SGD-2083), add in the culture medium of 100 μ M alkynyl fucosees (SGD-1887), or cultivated for two weeks in control medium (not adding the fucose analog).Growth medium is the MEM Eagle (LS174T) that is added with 10%FBS, be added with 10%FBS 50:50F12 and
RPMI (PC-3), be added with the RPMI (HL-60) of 10%FBS, is added with the IMDM of 10%FBS
, and the McCoy (PC-3) that is added with 10%FBS (Ramos).These cells are assessed the fucosylation of cell surface by FACS, cBR96 detects LewisY with antibody, and antibody SSEA-1 detects LewisX, and the palatelet-selectin part detects palatelet-selectin, and the AOL coagulation usually detects rock
The glycosylated aggregate level of algae.
Result:
The result of FACS assessment shows the level different (data do not show) of the cell surface protein fucosylation of different cell line.2-fluorine fucose (SGD-2083) is than alkynyl fucose (SGD-generally
1887) better protein fucosylation inhibitor.
In order further to assess the activity of these fucose analog, use and pass through at the fucose analog
There is lower the cultivation and pretreated tumor cell or use undressed tumor cell further to study in vivo.As described below tumor cell is implanted in every group of 10 mices.To LS174T, PC-3,
And Caki-1 cell line, will under 5x105 cell skin in 25% matrigel (Matrigel), be implanted in female nude mouse.To HL-60 and Ramos cell line, will under 5x106 cell skin, be implanted in female SCID Mice Body.For the mice of having implanted unprocessed tumor cell, provide conventional drinking water.To having implanted the mice through the pretreated tumor cell of 2-fluorine fucose (SGD-2083), provide the drinking water that is added with 20mM2-fluorine fucose (SGD-2083).Mice for having implanted through the pretreated tumor cell of alkynyl fucose (SGD-1887), provide conventional drinking water.Mice is not drunk and contains
The water that the alkynyl fucose is arranged.
Drinking the drinking water that contains 2-fluorine fucose after continuous three weeks, mice recovers to drink conventional drinking water, but with except the mice of Caki-1 tumor.The latter recovers conventional one week of drinking water.Drinking conventional water after one week, mice is divided into to two groups at random, 5 every group, respectively drink the drinking water or the conventional drinking water that are added with 20mM2-fluorine fucose.When tumor reaches about 1000mm3, put to death mice.
Referring to Fig. 8 A-E, through LS174T, PC-3 and the Caki-1 cell observation that 2-fluorine fucose (SGD-2083) is processed, to the tumor growth in body, suppress.HL-60 and Ramos cell are not observed the variation of tumor growth.To the Caki-1 cell, in the first processing phase, do not observe tumor growth and suppress, but recover after 2-fluorine fucose is processed to observe tumor growth mice, suppress.For other cell line, it seems that the tumor growth inhibition when gross tumor volume reaches 150mm3, start.The slower Caki-1 tumor of growing until the second processing phase of 2-fluorine fucose (SGD-2083) just reach this node.These results show that using the fucose analog to process can suppress tumor growth.
Research 3
In the 3rd research, implant without the pretreated tumor cell of fucose analog.By subcutaneous being implanted in female nude mouse of LS174T colon adenocarcinoma cell (cell of the 5x105 in 25% matrigel).In implantation, start to implanting, to give drinking water or the conventional drinking water that mice contains 50mM2-fluorine fucose (SGD-2083) in latter the 21st day in first 7 days.
Result
Referring to Fig. 8 F, the mice that gives 50mM2-fluorine fucose (SGD-2083) in drinking water demonstrates obvious tumor growth and suppresses, and the tumor meansigma methods that reaches is 110mm3, and the mice that gives by contrast conventional drinking water is 734mm3.In a word, these results show that giving the fucose analog can suppress tumor growth.
Embodiment 8: the tumor vaccine model
At the-21 days and the-7 days, female Balb/c mice was implanted 100 ten thousand A20 Mus lymphoma cells (through the radiation deactivation) by subcutaneous implantation and comes immune.Another group mice is not subject to any immunity.At the 0th day, all mices are the A20 cell of intravenous inoculation 1.5 hundred ten thousand or 500 ten thousand work all, at the-14 to+21 days, gives mice and contains the drinking water of 50mM2-fluorine fucose (SGD-2083) or give conventional drinking water.8 processed group are as follows:
1. without immunity, the A20 cell of 1.5 hundred ten thousand work, conventional drinking water
2. without immunity, the A20 cell of 500 ten thousand work, conventional drinking water
3. without immunity, the A20 cell of 1.5 hundred ten thousand work, contain the drinking water of 50mM SGD-2083
4. without immunity, the A20 cell of 500 ten thousand work, contain the drinking water of 50mM SGD-2083
5. through immunity, the A20 cell of 1.5 hundred ten thousand work, conventional drinking water
6. through immunity, the A20 cell of 500 ten thousand work, conventional drinking water
7. through immunity, the A20 cell of 1.5 hundred ten thousand work, contain the drinking water of 50mM SGD-2083
8. through immunity, the A20 cell of 500 ten thousand work, contain the drinking water of 50mM SGD-2083
Result
Referring to Fig. 9 A, shown research approach.Referring to Fig. 9 B, from 22-35 days, without the mice of immune-treated, from 22-35 days, die from the attack of A20 alive.The mice that the mice of accepting 2-fluorine fucose (SGD-2083) is drunk conventional drinking water survives a couple of days more.A20 cell with 500 ten thousand deactivations
Immunity and two mices of drinking conventional drinking water die from the attack of A20 alive.All are accepted immunity and drink the mice of the drinking water that contains 2-fluorine fucose (SGD-2083) until all still survivals during data acquisition.
The present invention is not limited to the scope of the specific embodiment as herein described.Except content described herein, those skilled in the art can understand various variation of the present invention by description and the accompanying drawing of front.The scope of appended claims is intended to comprise these variations.Unless be obvious from context, any step of the present invention, element, embodiment, feature or aspect can be used in combination mutually.All patent applications of quoting in the application, scientific publications, accession number etc. are all included in for all purposes by reference in full, just as figure, list separately each piece document.

Claims (59)

1. suppress the method for protein fucosylation in mammal, described method comprises the fucose analog that described mammal is given to effective dose, described fucose analog be selected from following molecule formula V or (VI) one of them or its biology acceptable salt or solvate:
Figure FDA00002816630200011
Chemical formula V or (VI) can be α or β anomer or corresponding aldose form separately wherein;
R 1, R 2, R 2a, R 3, R 3aAnd R 4Be selected from independently of one another following group :-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle) ,-OCH 2OC (O) alkyl ,-OCH 2OC (O) O alkyl ,-OCH 2OC (O) aryl ,-OCH 2OC (O) O aryl ,-OC (O) CH 2O (CH 2CH 2O) nCH 3,-OC (O) CH 2CH 2O (CH 2CH 2O) nCH 3,-O-tri--C 1-C 3Alkyl silicyl, – OC 1-C 10Alkyl and little electron withdraw group, wherein each n is the integer that independently is selected from 0-5;
R 5To be selected from the member: – CH of lower group 3,-CHX 2,-CH 2X, by halogen, replaced or unsubstituted-CH (X')-C 1-C 4Alkyl, by halogen, replaced or unsubstituted-CH (X')-C 2-C 4Thiazolinyl, by halogen, replaced or unsubstituted-CH (X')-C 2-C 4Alkynyl ,-CH=C (R 10) (R 11) ,-C (CH 3)=C (R 12) (R 13) ,-C (R 14)=C=C (R 15) (R 16), by methyl or halogen replace or unsubstituted-C 3Carbocyclic ring, by halogen methyl substituted or unsubstituted-CH (X')-C 3Carbocyclic ring, by halogen methyl substituted or unsubstituted C 3Heterocycle, by halogen methyl substituted or unsubstituted-CH (X')-C 3Heterocycle ,-CH 2N 3,-CH 2CH 2N 3And benzyloxymethyl, or R 5It is little electron withdraw group; Wherein
R 10Be hydrogen or by halogen, replaced or unsubstituted C 1-C 3Alkyl;
R 11C that replaced by halogen or unsubstituted 1-C 3Alkyl;
R 12Hydrogen, halogen or that by halogen, replaced or unsubstituted C 1-C 3Alkyl; And
R 13Hydrogen, or that by halogen, replaced or unsubstituted C 1-C 3Alkyl;
R 14Hydrogen or methyl;
R 15And R 16Be independently selected from hydrogen, methyl and halogen;
X is halogen; And
X' is halogen or hydrogen; And
Extraly, R 1, R 2, R 2a, R 3And R 3aBe chosen as separately hydrogen; Can be selected in the R on contiguous carbon atom 1, R 2, R 2a, R 3And R 3aIn two between above-mentioned contiguous carbon atom, combine and form two keys; And
Prerequisite is R 1, R 2, R 2a, R 3, R 3a, R 4, R 5In at least one is little electron withdraw group, or R 5Comprise halogen, unsaturated site, carbocyclic ring, heterocycle or azide, unless (i) R 2With R 2aBe hydrogen, (ii) R 3With R 3aBe hydrogen, (iii) R 1For hydrogen, (iv) between described contiguous carbon atom, there are two keys, or (v) R 5For benzyloxymethyl; And
The protein fucosylation amount that in wherein said mammal, the protein fucosylation does not give in the mammal of described fucose analog of molecular formula V or VI relatively reduces at least 10%.
2. the method for claim 1, is characterized in that, described fucose analog is selected from following molecular formula (I) or (II) or its biologically acceptable salt or solvate:
Figure FDA00002816630200021
Wherein:
Chemical formula (I) or (II) can be α or β anomer or corresponding aldose form separately;
R 1To R 4Be selected from independently of one another following group :-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle) ,-OCH 2OC (O) alkyl ,-OCH 2OC (O) O alkyl ,-OCH 2OC (O) aryl ,-OCH 2OC (O) O aryl ,-OC (O) CH 2O (CH 2CH 2O) nCH 3,-OC (O) CH 2CH 2O (CH 2CH 2O) nCH 3,-O-tri--C 1-C 3The alkyl silicyl ,-OC 1-C 10Alkyl, wherein each n is the integer that independently is selected from 0-5; And
R5 is selected from lower group :-C ≡ CH ,-C ≡ CCH 3,-CH 2C ≡ CH ,-C (O) OCH 3,-CH (OAc) CH 3,-CN ,-CH 2CN ,-CHX 2(wherein each X is F, Br or Cl) ,-CH 2X (wherein X is F, Br, Cl or I) and methyl oxirane.
3. method as claimed in claim 1 or 2, is characterized in that, described mammal has cancer.
4. method as claimed in claim 1 or 2, is characterized in that, described mammal has the autoimmune disorder.
5. method as claimed in claim 3, also comprise giving cancer associated antigen or its antigenicity fragment is usingd as immunogen.
6. method as claimed in claim 1 or 2, is characterized in that, R 1To R 4Be selected from independently of one another lower group :-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle) ,-OC (O) CH 2O (CH 2CH 2O) nCH 3With-OC (O) CH 2CH 2O (CH 2CH 2O) nCH 3And R 5Be selected from lower group :-C ≡ CH ,-C ≡ CCH 3,-CH 2C ≡ CH ,-C (O) OCH 3,-CH (OAc) CH 3,-CN ,-CH 2CN ,-CH 2X (wherein X is F, Br, Cl or I) and methyl oxirane.
7. method as claimed in claim 1 or 2, is characterized in that, R 1To R 4Be selected from independently of one another lower group :-OH-OC (O) H and-OC (O) C 1-C 10Alkyl.
8. method as claimed in claim 7, is characterized in that, R 1To R 4Be selected from independently of one another-OH is with – OAc.
9. method as claimed in claim 1 or 2, is characterized in that, R 5Be selected from-C ≡ CH and-C ≡ CCH 3.
10. method as claimed in claim 9, is characterized in that, R 5For-C ≡ CH.
11. method as claimed in claim 1 or 2, is characterized in that, R 5For-CH 2X, wherein X is F, Br, Cl or I.
12. method as claimed in claim 11, is characterized in that, R 5For-CH 2X, wherein X is F.
13. method as claimed in claim 11, is characterized in that, R 5For-CH 2X, wherein X is Br.
14. method as claimed in claim 1 or 2, is characterized in that, R 5For-CHX 2, wherein X is F.
15. method as claimed in claim 1 or 2, is characterized in that, R 5For-CHX 2, wherein X is F.
16. method as claimed in claim 1 or 2, is characterized in that, R 5For-CHF 2And R 1To R 4For OAc.
17. method as claimed in claim 1 or 2, is characterized in that, R 5Wei – CH 2Br and R 1-R 4For-OAc.
18. method as claimed in claim 1 or 2, is characterized in that, R 5For-C ≡ CH and R 1-R 4For-OAc.
19. the method for claim 1, is characterized in that, described fucose analog is selected from molecular formula (III) or (IV) biologically acceptable salt or solvate of one of them or its:
Chemical formula (III) or (IV) can be α or β anomer or corresponding aldose form separately wherein;
R 1To R 4Be selected from independently of one another: fluorine, chlorine, OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle) ,-OCH 2OC (O) alkyl ,-OCH 2OC (O) O alkyl ,-OCH 2OC (O) aryl ,-OCH 2OC (O) O aryl ,-OC (O) CH 2O (CH 2CH 2O) nCH 3,-OC (O) CH 2CH 2O (CH 2CH 2O) nCH 3,-O-tri--C 1-C 3Alkyl silicyl and-OC 1-C 10Alkyl, wherein each n independently is selected from the integer of 0-5; And
R 2aTo R 3aBe selected from independently of one another H, F and Cl;
R 5Be selected from lower group :-CH 3,-CHF 2,-CH=C=CH 2,-C ≡ CH ,-C ≡ CCH 3,-CH 2C ≡ CH ,-C (O) OCH 3,-CH (OAc) CH 3,-CN ,-CH 2CN ,-CH 2X (wherein X is F, Br, Cl or I) and methyl oxirane.
20. method as claimed in claim 19, is characterized in that, R 1For F.
21. method as claimed in claim 19, is characterized in that, R 2For F.
22. method as claimed in claim 19, is characterized in that, R 3For F.
23. method as claimed in claim 19, is characterized in that, R 2With R 2aBe respectively F.
24. method as claimed in claim 19, is characterized in that, R 1With R 2Be respectively F.
25. method as claimed in claim 19, is characterized in that, R 2aWith R 3aBe respectively hydrogen.
26. method as claimed in claim 19, is characterized in that, R 5Be selected from lower group :-CH 3,-CH 2CH 3,-C ≡ CH ,-CH 2C ≡ CH ,-CH=CHCH 3,-cyclopropyl ,-oxirane, methyl substituted-oxirane ,-CH 2F ,-CH 2Cl ,-CH 2Br ,-CH 2I ,-CHF 2,-CH=C=CH 2,-CH 2N 3With-CH 2CH 2N 3.
27. method as claimed in claim 19, is characterized in that, described little electron withdraw group is selected from lower group: fluorine, chlorine, bromine ,-CHF 2,-CH=C=CH 2,-C ≡ CH ,-C ≡ CCH 3,-CH 2C ≡ CH ,-CO 2H ,-C (O) OC 1-C 4Alkyl ,-CH (OAc) CH 3,-CN ,-CH 2CN ,-CH 2X (wherein X is F, Br, Cl or I) and methyl oxirane.
28. method as claimed in claim 19, is characterized in that, R 5For-C ≡ CH.
29. method as claimed in claim 19, is characterized in that, R 5For-CH 2X, wherein X is F, Br, Cl or I.
30. method as claimed in claim 29, is characterized in that, R 5For-CH 2X, wherein X is F.
31. method as claimed in claim 29, is characterized in that, R 5For-CH 2X, wherein X is Br.
32. method as claimed in claim 19, is characterized in that, R 5For-CHX 2, wherein X is F.
33. method as claimed in claim 25, is characterized in that, R 5For-CHX 2, wherein X is F.
34. method as claimed in claim 19, is characterized in that, R 5For-CHF 2, and R 1-R 4For-OAc.
35. method as claimed in claim 19, is characterized in that, R 5For-CH 2Br, and R 1-R 4For-OAc.
36. method as claimed in claim 19, is characterized in that, R 5For-C ≡ CH, and R 1-R 4For-OAc.
37. method as claimed in claim 19, is characterized in that, R 1, R 2, R 2a, R 3, R 3aWith R 4Wherein at least two is little electron withdraw group.
38. method as claimed in claim 19, is characterized in that, described fucose analog is selected from table 1,2,3 compound.
39. preparation is for giving mammiferous pharmaceutical composition, described compositions comprises the fucose analog of effective dose, its be selected from following molecule formula V or (VI) one of them or its biology acceptable salt or solvate:
Figure FDA00002816630200071
Figure FDA00002816630200081
Chemical formula V or (VI) can be α or β anomer or corresponding aldose form separately wherein;
R 1, R 2, R 2a, R 3, R 3a, R 4Be selected from independently of one another lower group :-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle) ,-OCH 2OC (O) alkyl ,-OCH 2OC (O) O alkyl ,-OCH 2OC (O) aryl ,-OCH 2OC (O) O aryl ,-OC (O) CH 2O (CH 2CH 2O) nCH 3,-OC (O) CH 2CH 2O (CH 2CH 2O) nCH 3,-O-tri--C 1-C 3The alkyl silicyl ,-OC 1-C 10Alkyl, and little electron withdraw group, wherein each n is the integer that independently is selected from 0-5;
R 5Be selected from lower group :-CH 3,-CHX 2,-CH 2X, that replaced by halogen or unsubstituted-CH (X ')-C 1-C 4Alkyl, that replaced by halogen or unsubstituted-CH (X ')-C 2-C 4Thiazolinyl, that replaced by halogen or unsubstituted-CH (X ')-C 2-C 4Alkynyl ,-CH=C (R 10) (R 11) ,-C (CH 3)=C (R 12) (R 13) ,-C (R 14)=C=C (R 15) (R 16), replace or unsubstituted-C by methyl or halogen 3Carbocyclic ring, by methyl or halogen, replaced or unsubstituted-CH (X ')-C 3Carbocyclic ring, replace or the unsubstituted C by methyl or halogen 3Heterocycle, by methyl or halogen, replaced or unsubstituted-CH (X ')-C 3Heterocycle ,-CH 2N 3,-CH 2CH 2N 3, and benzyloxymethyl, or R 5It is little electron withdraw group; Wherein
R 10Be hydrogen or by halogen, replaced or unsubstituted C 1-C 3Alkyl;
R 11By halogen, replaced or unsubstituted C 1-C 3Alkyl;
R 12Hydrogen, halogen or replaced by halogen or unsubstituted C 1-C 3Alkyl;
R 13Be hydrogen, or by halogen, replaced or unsubstituted C 1-C 3Alkyl;
R 14Hydrogen or methyl;
R 15With R 16Be independently selected from hydrogen, methyl or halogen;
X is halogen; And
X ' is halogen or hydrogen; And
Extraly, R 1, R 2, R 2a, R 3With R 3aBe chosen as separately hydrogen; The optional R that is positioned on contiguous carbon atom 1, R 2, R 2a, R 3With R 3aWherein two combine and between described contiguous carbon atom, form two keys; And
Prerequisite is, R 1, R 2, R 2a, R 3, R 3a, R 4With R 5In at least one is little electron withdraw group, or R 5Comprise halogen, unsaturated site, carbocyclic ring, heterocycle or azide, unless (ⅰ) R 2With R 2aBe hydrogen, (ⅱ) R 3With R 3aBe hydrogen, (ⅲ) R 1For hydrogen, (ⅳ) between described contiguous carbon atom, there are two keys, or (ⅴ) R 5For benzyloxymethyl; And
The protein fucosylation amount that protein fucosylation in wherein said mammal does not give in the animal of fucose analog shown in molecular formula V or VI relatively has reduction.
40. a kind of pharmaceutical composition as claimed in claim 39, is characterized in that,
R 1To R 4Be selected from independently of one another lower group :-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle) ,-OCH 2OC (O) alkyl ,-OCH 2OC (O) O alkyl ,-OCH 2OC (O) aryl ,-OCH 2OC (O) O aryl ,-OC (O) CH 2O (CH 2CH 2O) nCH 3,-OC (O) CH 2CH 2O (CH 2CH 2O) nCH 3,-O-tri--C 1-C 3The alkyl silicyl ,-OC 1-C 10Alkyl, wherein each n is the integer that is independently selected from 0-5; And
R 5Be selected from lower group :-C ≡ CH ,-C ≡ CCH 3,-CH 2C ≡ CH ,-C (O) OCH 3,-CH (OAc) CH 3,-CN ,-CH 2CN ,-CH 2X (wherein each X is F, Br, Cl or I) ,-CH 2X (wherein X is F, Br, Cl or I) and methyl oxirane.
41. pharmaceutical composition as claimed in claim 39, is characterized in that,
R 1To R 4Be selected from independently of one another lower group :-OH ,-OC (O) H ,-OC (O) C 1-C 10Alkyl ,-OC (O) C 2-C 10Thiazolinyl ,-OC (O) C 2-C 10Alkynyl ,-OC (O) aryl ,-OC (O) heterocycle ,-OC (O) C 1-C 10Alkylidene (aryl) ,-OC (O) C 2-C 10Alkenylene (aryl) ,-OC (O) C 2-C 10Alkynylene (aryl) ,-OC (O) C 1-C 10Alkylidene (heterocycle) ,-OC (O) C 2-C 10Alkenylene (heterocycle) ,-OC (O) C 2-C 10Alkynylene (heterocycle) ,-OCH 2OC (O) alkyl ,-OCH 2OC (O) O alkyl ,-OCH 2OC (O) aryl ,-OCH 2OC (O) O aryl ,-OC (O) CH 2O (CH 2CH 2O) nCH 3,-OC (O) CH 2CH 2O (CH 2CH 2O) nCH 3,-O-tri--C 1-C 3The alkyl silicyl ,-OC 1-C 10Alkyl, wherein each n is the symptom that is independently selected from 0-5; And R 2For F, R 2aWith R 3aBe respectively H, R 5For-CH 3.
42. compositions as claimed in claim 39, is characterized in that, R 1For F.
43. compositions as claimed in claim 39, is characterized in that, R 2For F.
44. compositions as claimed in claim 39, is characterized in that, R 3For F.
45. compositions as claimed in claim 39, is characterized in that, R 2With R 2aBe respectively F.
46. method as claimed in claim 39, is characterized in that, R 1With R 2Be respectively F.
47. compositions as claimed in claim 19, is characterized in that, R 2aWith R 3aBe respectively hydrogen.
48. compositions as claimed in claim 39, is characterized in that, R 5Be selected from lower group :-CH 3,-CH 2CH 3,-C ≡ CH ,-CH 2C ≡ CH ,-CH=CHCH 3,-cyclopropane ,-oxirane, methyl substituted-oxirane ,-CH 2F ,-CH 2Cl ,-CH 2Br ,-CH 2I ,-CHF 2,-CH=C=CH 2,-CH 2N 3With-CH 2CH 2N 3.
49. compositions as claimed in claim 39, is characterized in that, described little electron withdraw group is selected from lower group: fluorine, chlorine, bromine ,-CHF 2,-CH=C=CH 2,-C ≡ CH ,-C ≡ CCH 3,-CH 2C ≡ CH ,-CO 2H ,-C (O) OC 1-C 4Alkyl ,-CH (OAc) CH 3,-CN ,-CH 2CN ,-CH 2X (wherein X is F, Br, Cl or I) and methyl oxirane.
50. compositions as claimed in claim 39, is characterized in that, R 5For-C ≡ CH.
51. compositions as claimed in claim 39, is characterized in that, R 5For-CH 2X, wherein X is F, Br, Cl or I.
52. compositions as claimed in claim 39, is characterized in that, R 5For-CH 2X, wherein X is F.
53. compositions as claimed in claim 39, is characterized in that, R 5For-CH 2X, wherein X is Br.
54. compositions as claimed in claim 39, is characterized in that, R 5For-CHX 2, wherein X is F.
55. compositions as claimed in claim 39, is characterized in that, R 5For-CHX 2, wherein X is F.
56. compositions as claimed in claim 39, is characterized in that, R 5For-CHF 2, and R 1-R 4For-OAc.
57. compositions as claimed in claim 39, is characterized in that, R 5For-CH 2Br, and R 1-R 4For-OAc.
58. compositions as claimed in claim 39, is characterized in that, R 5For-C ≡ CH, and R 1-R 4For-OAc.
59. compositions as claimed in claim 19, is characterized in that, R 1, R 2, R 2a, R 3, R 3aWith R 4In at least two be little electron withdraw group.
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